Fluorescent silica-based nanoparticles

ABSTRACT

The present invention provides a fluorescent silica-based nanoparticle that allows for precise detection, characterization, monitoring and treatment of a disease such as cancer. The nanoparticle has a range of diameters including between about 0.1 nm and about 100 nm, between about 0.5 nm and about 50 nm, between about 1 nm and about 25 nm, between about 1 nm and about 15 nm, or between about 1 nm and about 8 nm. The nanoparticle has a fluorescent compound positioned within the nanoparticle, and has greater brightness and fluorescent quantum yield than the free fluorescent compound. The nanoparticle also exhibits high biostability and biocompatibility. To facilitate efficient urinary excretion of the nanoparticle, it may be coated with an organic polymer, such as poly(ethylene glycol) (PEG). The small size of the nanoparticle, the silica base and the organic polymer coating minimizes the toxicity of the nanoparticle when administered in vivo. In order to target a specific cell type, the nanoparticle may further be conjugated to a ligand, which is capable of binding to a cellular component associated with the specific cell type, such as a tumor marker. In one embodiment, a therapeutic agent may be attached to the nanoparticle. To permit the nanoparticle to be detectable by not only optical fluorescence imaging, but also other imaging techniques, such as positron emission tomography (PET), single photon emission computed tomography (SPECT), computerized tomography (CT), bioluminescence imaging, and magnetic resonance imaging (MM), radionuclides/radiometals or paramagnetic ions may be conjugated to the nanoparticle.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/381,209, filed Sep. 27, 2012, which is a US national phase entry ofInternational Application No. PCT/US10/40994, filed Jul. 2, 2010, whichclaims priority to U.S. Provisional Application Nos. 61/312,827, filedMar. 11, 2010, and 61/222,851, filed Jul. 2, 2009, the contents of whichare hereby incorporated by reference in their entireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under grant numbersCA086438, CA083084, CA008748, and RR024996 awarded by NationalInstitutes of Health. The government has certain rights in theinvention.

FIELD OF THE INVENTION

The present invention relates to fluorescent silica-based nanoparticles,and methods of using the nanoparticles to detect, diagnose, or treatdiseases such as cancer.

BACKGROUND OF THE INVENTION

Early tumor detection and treatment selection is paramount to achievingtherapeutic success and long-term survival rates. At its early stage,many cancers are localized and can be treated surgically. However,well-defined tumor margins are often difficult to visualize with currentimaging techniques. This has led to a disproportionate number ofinvasive biopsies. Highly-specific, molecular-targeted probes are neededfor the early detection of molecular differences between normal andtumor cells, such as cancer-specific alterations in receptor expressionlevels. When combined with high-resolution imaging techniques, specificmolecular-targeted probes will greatly improve detection sensitivity,facilitating characterization, monitoring and treatment of cancer.

Current fluorescence imaging probes typically consist of singleconventional fluorophore (e.g., organic dyes, fluorescent proteins),fluorescent proteins (e.g., GFP) and semiconductor quantum dots(Q-dots). Single fluorophores are usually not stable and have limitedbrightness for imaging. Similar to dyes, the fluorescent proteins tendto exhibit excited state interactions which can lead to stochasticblinking, quenching and photobleaching. Q-dots are generally made fromheavy metal ions such as Pb²⁺ or Cd²⁺ and, therefore, are toxic. Burnset al. “Fluorescent core-shell silica nanoparticles: towards “Lab on aParticle” architectures for nanobiotechnology”, Chem. Soc. Rev., 2006,35, 1028-1042.

Fluorescent nanoparticles having an electrically conducting shell and asilica core are known and have utility in modulated delivery of atherapeutic agent. U.S. Pat. Nos. 6,344,272, and 6,428,811. Ashortcoming of existing fluorescent nanoparticles is their limitedbrightness and their low detectability as fluorescent probes indispersed systems.

The present multifunctional fluorescent silica-based nanoparticles offermany advantages over other fluorescent probes. The nanoparticles arenon-toxic, and have excellent photophysical properties (includingfluorescent efficiency and photostability), high biocompatibility, andunique pharmacokinetics for molecular diagnostics and therapeutics. Thenanoparticles are relatively small in size, and have a surface PEGcoating that offers excellent renal clearance. The fluorescentnanoparticles of the present invention contain a fluorescent core andsilica shell. The core-shell architectures, the great surface area anddiverse surface chemistry of the nanoparticle permit multiplefunctionalities simultaneously delivered to a target cell. For example,the nanoparticle can be functionalized with targeting moieties, contrastagents for medical imaging, therapeutic agents, or other agents. Thetargeting moieties on the surface of the nanoparticle may be tumorligands, which, when combined with nanoparticle-conjugated therapeuticagents, makes the nanoparticle an ideal vehicle for targeting andpotentially treating cancer. Webster et al. Optical calcium sensors:development of a generic method for their introduction to the cell usingconjugated cell penetrating peptides. Analyst, 2005; 130:163-70. Thesilica-based nanoparticle may be labeled with contrast agents for PET,SPECT, CT, MRI, and optical imaging.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a fluorescentsilica-based nanoparticle comprising a silica-based core having afluorescent compound positioned within the silica-based core; a silicashell surrounding at least a portion of the core; an organic polymerattached to the nanoparticle; from about 1 to about 20 ligands attachedto the nanoparticle; and a contrast agent or a chelate attached to thenanoparticle. The diameter of the nanoparticle ranges from about 1 nm toabout 25 nm, or from about 1 nm to about 8 nm. The organic polymers thatmay be attached to the nanoparticle include poly(ethylene glycol) (PEG),polylactate, polylactic acids, sugars, lipids, polyglutamic acid (PGA),polyglycolic acid, poly(lactic-co-glycolic acid) (PLGA), Polyvinylacetate (PVA), or the combinations thereof.

The ligand may be capable of binding to at least one cellular component,such as a tumor marker. The number of ligands attached to thenanoparticle may also range from about 1 to about 10. Examples of theligand include peptide, protein, biopolymer, synthetic polymer, antigen,antibody, microorganism, virus, receptor, hapten, enzyme, hormone,chemical compound, pathogen, toxin, surface modifier, or combinationsthereof. Peptides such as tripeptide RGD, cyclic peptide cRGD,octreotate, EPPT1 and peptide analogs of alpha-MSH are encompassed bythe present invention. Any linear, cyclic or branched peptide containingthe RGD sequence is within the scope of the present invention.

A contrast agent, such as a radionuclide including ⁸⁹Zr, ⁶⁴Cu, ⁶⁸Ga,⁸⁶Y, ¹²⁴I and ¹⁷⁷Lu, may be attached to the nanoparticle. Alternatively,the nanoparticle is attached to a chelate, for example, DFO, DOTA, TETAand DTPA, that is adapted to bind a radionuclide. The nanoparticle ofthe present invention may be detected by positron emission tomography(PET), single photon emission computed tomography (SPECT), computerizedtomography (CT), magnetic resonance imaging (MRI), optical imaging (suchas fluorescence imaging including near-infrared fluorescence (NIRF)imaging), bioluminescence imaging, or combinations thereof.

A therapeutic agent may be attached to the nanoparticle. The therapeuticagents include antibiotics, antimicrobials, antiproliferatives,antineoplastics, antioxidants, endothelial cell growth factors, thrombininhibitors, immunosuppressants, anti-platelet aggregation agents,collagen synthesis inhibitors, therapeutic antibodies, nitric oxidedonors, antisense oligonucleotides, wound healing agents, therapeuticgene transfer constructs, extracellular matrix components,vasodialators, thrombolytics, anti-metabolites, growth factor agonists,antimitotics, statin, steroids, steroidal and non-steroidalanti-inflammatory agents, angiotensin converting enzyme (ACE)inhibitors, free radical scavengers, PPAR-gamma agonists, smallinterfering RNA (siRNA), microRNA, and anti-cancer chemotherapeuticagents. The therapeutic agents encompassed by the present invention alsoinclude radionuclides, for example, ⁹⁰Y, ¹³¹I and ¹⁷⁷Lu. The therapeuticagent may be radiolabeled, such as labeled by binding to radiofluorine¹⁸F.

After administration of the nanoparticle to a subject, blood residencehalf-time of the nanoparticle may range from about 2 hours to about 25hours, from about 3 hours to about 15 hours, or from about 4 hours toabout 10 hours. Tumor residence half-time of the nanoparticle afteradministration of the nanoparticle to a subject may range from about 5hours to about 5 days, from about 10 hours to about 4 days, or fromabout 15 hours to about 3.5 days. The ratio of tumor residence half-timeto blood residence half-time of the nanoparticle after administration ofthe nanoparticle to a subject may range from about 2 to about 30, fromabout 3 to about 20, or from about 4 to about 15. Renal clearance of thenanoparticle after administration of the nanoparticle to a subject mayrange from about 10% ID (initial dose) to about 100% ID in about 24hours, from about 30% ID to about 80% ID in about 24 hours, or fromabout 40% ID to about 70% ID in about 24 hours. In one embodiment, afterthe nanoparticle is administered to a subject, blood residence half-timeof the nanoparticle ranges from about 2 hours to about 25 hours, tumorresidence half-time of the nanoparticle ranges from about 5 hours toabout 5 days, and renal clearance of the nanoparticle ranges from about30% ID to about 80% ID in about 24 hours.

When the nanoparticles in the amount of about 100 times of the humandose equivalent are administered to a subject, substantially no anemia,weight loss, agitation, increased respiration, GI disturbance, abnormalbehavior, neurological dysfunction, abnormalities in hematology,abnormalities in clinical chemistries, drug-related lesions in organpathology, mortality, or combinations thereof, is observed in thesubject in about 10 to about 14 days.

Multivalency enhancement of the nanoparticle may range from about 2 foldto about 4 fold.

The present invention also provides a fluorescent silica-basednanoparticle comprising a silica-based core comprising a fluorescentcompound positioned within the silica-based core; a silica shellsurrounding at least a portion of the core; an organic polymer attachedto the nanoparticle; and a ligand attached to the nanoparticle, whereinthe nanoparticle has a diameter between about 1 nm and about 15 nm.After administration of the nanoparticle to a subject, blood residencehalf-time of the nanoparticle ranges from about 2 hours to about 25hours, tumor residence half-time of the nanoparticle ranges from about 5hours to about 5 days, and renal clearance of the nanoparticle rangesfrom about 30% ID to about 80% ID in about 24 hours. The number ofligands attached to the nanoparticle may range from about 1 to about 20,or from about 1 to about 10. The diameter of the nanoparticle may bebetween about 1 nm and about 8 nm. A contrast agent, such as aradionuclide, may be attached to the nanoparticle. Alternatively, achelate may be attached to the nanoparticle. The nanoparticle may bedetected by PET, SPECT, CT, MRI, optical imaging, bioluminescenceimaging, or combinations thereof. A therapeutic agent may be attached tothe nanoparticle. After administration of the nanoparticle to a subject,blood residence half-time of the nanoparticle may also range from about3 hours to about 15 hours, or from about 4 hours to about 10 hours.Tumor residence half-time of the nanoparticle after administration ofthe nanoparticle to a subject may also range from about 10 hours toabout 4 days, or from about 15 hours to about 3.5 days. The ratio oftumor residence half-time to blood residence half-time of thenanoparticle after administration of the nanoparticle to a subject mayrange from about 2 to about 30, from about 3 to about 20, or from about4 to about 15. Renal clearance of the nanoparticle may also ranges fromabout 40% ID to about 70% ID in about 24 hours after administration ofthe nanoparticle to a subject.

Also provided in the present invention is a fluorescent silica-basednanoparticle comprising a silica-based core comprising a fluorescentcompound positioned within the silica-based core; a silica shellsurrounding at least a portion of the core; an organic polymer attachedto the nanoparticle; and a ligand attached to the nanoparticle, whereinthe nanoparticle has a diameter between about 1 nm and about 8 nm. Afteradministration of the nanoparticle to a subject, the ratio of tumorresidence half-time to blood residence half-time of the nanoparticleranges from about 2 to about 30, and renal clearance of the nanoparticleranges from about 30% ID to about 80% ID in about 24 hours.

The present invention further provides a method for detecting acomponent of a cell comprising the steps of: (a) contacting the cellwith a fluorescent silica-based nanoparticle comprising a silica-basedcore comprising a fluorescent compound positioned within thesilica-based core; a silica shell surrounding at least a portion of thecore; an organic polymer attached to the nanoparticle; from about 1 toabout 20 ligands attached to the nanoparticle; and a contrast agent or achelate attached to the nanoparticle; and (b) monitoring the binding ofthe nanoparticle to the cell or a cellular component by at least oneimaging technique.

The present invention further provides a method for targeting a tumorcell comprising administering to a cancer patient an effective amount ofa fluorescent silica-based nanoparticle comprising a silica-based corecomprising a fluorescent compound positioned within the silica-basedcore; a silica shell surrounding at least a portion of the core; anorganic polymer attached to the nanoparticle; a ligand attached to thenanoparticle and capable of binding a tumor marker; and at least onetherapeutic agent. The nanoparticle may be radiolabeled. Thenanoparticle may be administered to the patient by, but not restrictedto, the following routes: oral, intravenous, nasal, subcutaneous, local,intramuscular or transdermal.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows a dynamic light scattering (DLS) plot (number average) ofparticle size for bare silica (gray) and PEG-coated (black)Cy5-containing silica nanoparticles.

FIG. 1B shows in vivo imaging of spectrally demixed Cy5 particlefluorescence (pseudocolor) overlaid on visible light imaging of nudemice 45 min post-injection with bare silica nanoparticles.

FIG. 1C shows in vivo imaging of spectrally demixed Cy5 particlefluorescence (pseudocolor) overlaid on visible light imaging of nudemice 45 min post-injection with PEG-ylated Cy5 nanoparticles.

FIG. 1D shows in vivo biodistribution study using co-registered PET-CT.Upper row is serial co-registered PET-CT image 24-hr after injection of¹²⁴I-labeled PEG coated nanoparticle, flanked by the independentlyacquired microCT and microPET scans. Lower row is serial microPETimaging.

FIG. 2A shows fluorescence correlation spectroscopy (FCS) data andsingle exponential fits for Cy5 dye (light gray), 3.3±0.06 nm diameter(dark gray, mean±standard deviation, n=9) and 6.0±0.1 nm diameter(black, mean±standard deviation, n=6) Cy5-containing PEG-coatednanoparticles showing the differences in diffusion time resulting fromthe different hydrodynamic sizes of the different species.

FIG. 2B shows absorption and emission spectra of Cy5 dye (light gray),3.3 nm diameter (dark gray) and 6.0 nm diameter (black) PEG-coatednanoparticles.

FIG. 2C shows relative brightness comparison of free dye (light gray)with 3.3 nm (dark gray) and 6.0 nm diameter (black) nanoparticles,measured as count rate per molecule/particle as determined from the FCScurves.

FIG. 2D shows photobleaching data for Cy5 dye (light gray), 3.3 nmdiameter (dark gray), and 6.0 nm diameter (black) PEG-coatednanoparticles under ˜3.5 mW laser excitation.

FIG. 3A shows percent of initial particle dose (% ID) retained by blood(black) and tissues: liver (light gray), lung (mid-low gray), spleen(midgray), and kidney (mid-high gray) for 6.0 nm diameter nanoparticlesat various time points from 10 min to 48 h post-injection (n=3 mice,mean±standard deviation).

FIG. 3B shows plot of retained particle concentration for 3.3 nm (lightgray) and 6.0 nm (black) diameter nanoparticles and the associatedlogarithmic decay fits and half-lives.

FIG. 3C shows plot of estimated particle excretion for 3.3 nm (lightgray) and 6.0 nm (black) diameter nanoparticles and the associatedlogarithmic fits and half-lives (mean±standard deviation, n=9 (threemice per time point)).

FIGS. 4A-4C show in vivo biodistribution of the nanoparticles innon-tumor-bearing and tumor-bearing mice with subcutaneous C6xenografts. (FIG. 4A) Bare silica particles; (FIGS. 4B-4C) PEGylated RGDparticles.

FIGS. 5A-5B show total specific binding data for cRGD- and PEG-ylateddots (i.e., nanoparticles) using flow cytometry in the Cy5 channel as afunction of time (FIG. 5A) and particle concentration (FIG. 5B).

FIGS. 6A-6C show multimodal C dot design for α_(v)β₃-integrin targetingand characterization.

FIG. 6A. Schematic representation of the ¹²⁴I-cRGDY-PEG-ylatedcore-shell silica nanoparticle with surface-bearing radiolabels andpeptides and core-containing reactive dye molecules (insets).

FIG. 6B. FCS results and single exponential fits for measurements of Cy5dyes in solution (black), PEGcoated (PEG-dot, red), and PEG-coated,cRGDY-labeled dots (blue, underneath red data set) showing diffusiontime differences as a result of varying hydrodynamic sizes.

FIG. 6C. Hydrodynamic sizes (mean±s.d., n=15), and relative brightnesscomparisons of the free dye with PEG-coated dots and cRGDY-PEG dotsderived from the FCS curves, along with the corresponding dye andparticle concentrations.

FIG. 7 shows purification and quality control of ¹²⁴I-RGDY-PEG-dotsusing size exclusion column chromatography. Radioactivity (right column)of ¹²⁴I-RGDdots and ¹²⁴I-PEG-dots detected by γ-counting andcorresponding fluorescence signal intensity (Cy5, left column) of¹²⁴I-RGDY-PEG-dots and ¹²⁴I-PEG-dots in each eluted fraction.

FIGS. 8A-8D show competitive integrin receptor binding studies with¹²⁴I-cRGDY-PEG-dots, cRGDY peptide, and anti-α_(v)β₃ antibody using twocell types.

FIG. 8A. High affinity and specific binding of ¹²⁴I-cRGDY-PEG-dots toM21 cells by γ-counting. Inset shows Scatchard analysis of binding dataplotting the ratio of the concentration receptor-bound (B) to unbound(or free, F) radioligand, or bound-to-free ratio, B/F, versus thereceptor-bound receptor concentration, B; the slope corresponds to thedissociation constant, Kd.

FIG. 8B. α_(v)β₃-integrin receptor blocking of M21 cells using flowcytometry and excess unradiolabeled cRGD or anti-α_(v)β₃ antibody priorto incubation with cRGDY-PEG-dots.

FIG. 8C. Specific binding of cRGDY-PEG-dots to M21 as against M21L cellslacking surface integrin expression using flow cytometry.

FIG. 8D. Specific binding of cRGDY-PEG-dots to HUVEC cells by flowcytometry. Each bar represents mean±s.d. of three replicates.

FIGS. 9A-9D show pharmacokinetics and excretion profiles of the targetedand non-targeted particle probes.

FIG. 9A. Biodistribution of ¹²⁴I-cRGDY-PEG-dots in M21 tumor-bearingmice at various times from 4 to 168 h p.i. The inset shows arepresentative plot of these data for blood to determine the residencehalf-time (T_(1/2)).

FIG. 9B. Biodistribution of ¹²⁴I-PEG-dots from 4 to 96 h postinjection.

FIG. 9C. Clearance profile of urine samples collected up to 168 hr p.i.of unradiolabeled cRGDY-PEG-dots (n=3 mice, mean±s.d.).

FIG. 9D. Corresponding cumulative % ID/g for feces at intervals up to168 hr p.i. (n=4 mice). For biodistribution studies, bars represent themean±s.d.

FIGS. 10A-10B show acute toxicity testing results.

FIG. 10A. Representative H&E stained liver at 400× (upper frames) andstained kidneys at 200× (lower frames). Mice were treated with a singledose of either non-radiolabeled ¹²⁷I-RGDY-PEG-dots or ¹²⁷I-PEG-coateddots (control vehicle) via intravenous injection and organs collected 14days later.

FIG. 10B. Average daily weights for each treatment group of the toxicitystudy. Scale bar in FIG. 10A corresponds to 100 μm.

FIGS. 11A-11D show serial in vivo PET imaging of tumor-selectivetargeting.

FIG. 11A. Representative whole-body coronal microPET images at 4 hrsp.i. demonstrating M21 (left, arrow) and M21L (middle, arrow) tumoruptakes of 3.6 and 0.7% ID/g, respectively, and enhanced M21 tumorcontrast at 24 hrs (right).

FIG. 11B. In vivo uptake of ¹²⁴I-cRGDY-PEG-dots in α_(v)β₃integrin-overexpressing M21 (black, n=7 mice) and non-expressing M21L(light gray, n=5 mice) tumors and ¹²⁴I-PEG-dots in M21 tumors (darkgray, n=5).

FIG. 11C. M21 tumor-to-muscle ratios for ¹²⁴I-cRGDY-PEG-dots (black) and¹²⁴I-PEG-dots (gray).

FIG. 11D. Correlation of in vivo and ex-vivo M21 tumor uptakes of cRGDYlabeled and unlabeled probes. Each bar represents the mean±s.d.

FIGS. 12A-12B show nodal mapping using multi-scale near-infrared opticalfluorescence imaging.

FIG. 12A. Whole body fluorescence imaging of the tumor site (T) anddraining inguinal (ILN) and axillary (ALN) nodes and communicatinglymphatics channels (bar, LC) 1-hr p.i. in a surgically-exposed livinganimal.

FIG. 12B. Corresponding co-registered white-light and high-resolutionfluorescence images (upper row) and fluorescence images only (lower row)revealing nodal infrastructure of local and distant nodes, includinghigh endothelial venules (HEV). The larger scale bar in (b) correspondsto 500 μm.

FIG. 13A shows the experimental setup of using spontaneous miniswinemelanoma model for mapping lymph node basins and regional lymphaticsdraining the site of a known primary melanoma tumor.

FIG. 13B shows small field-of-view PET image 5 minutes after subdermalinjection of multimodal particles (¹²⁴I-RGD-PEG-dots) about the tumorsite.

FIG. 14A shows whole-body dynamic ¹⁸F-fluorodeoxyglucose (¹⁸F-FDG) PETscan demonstrating sagittal, coronal, and axial images through the siteof nodal disease in the neck.

FIG. 14B shows fused ¹⁸F-FDG PET-CT scans demonstrating sagittal,coronal, and axial images through the site of nodal disease in the neck.

FIG. 14C shows the whole body miniswine image.

FIGS. 15A-15C show the same image sets as in FIGS. 14A-14C, but at thelevel of the primary melanoma lesion, adjacent to the spine on the upperback.

FIG. 16A shows high resolution dynamic PET images following subdermal,4-quadrant injection of ¹²⁴I-RGD-PEG-dots about the tumor site over a 1hour time period.

FIG. 16B shows fused PET-CT images following subdermal, 4-quadrantinjection of ¹²⁴I-RGD-PEG-dots about the tumor site over a 1 hour timeperiod.

FIG. 16C shows Cy5 imaging (top image), the resected node (second to topimage), and H&E staining (lower two images).

FIG. 17 shows a scheme for a nanoparticle with a fluorescent dye withinthe core and a PEG surface-coating. The nanoparticle is decorated withtriple bonds for subsequent “click chemistry” with both DFO andTyr3-octreotate functionalized with azide groups.

FIG. 18 shows structures of PEG derivative. Standard chemical reactionsare used for the production of the functionalized PEG with triple bonds,which will then be covalently attached to the nanoparticle via thesilane group.

FIG. 19 shows structures of DFO derivatives.

FIG. 20A shows structures of Tyr3-octreotate.

FIG. 20B shows synthesis of the azide-containing acid for incorporationinto Tyr3-Octreotate.

FIG. 21A shows a scheme of the production of functionalized nanoparticlewith an NIR fluorescent dye within its core, a PEG surface-coating, DFOchelates and Tyr3-octreotate.

FIG. 21B shows a scheme of the production of a multimodality⁸⁹Zr-labeled nanoparticle (PET and fluorescence) decorated withTyr3-octreotate.

FIG. 22 shows microscopic images demonstrating co-localization betweencRGF-PEG-nanoparticles and lysotracker red in the endocytotic pathway.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a fluorescent silica-based nanoparticlethat allows for precise detection, characterization, monitoring andtreatment of a disease such as cancer. The nanoparticle has a range ofdiameters including between about 0.1 nm and about 100 nm, between about0.5 nm and about 50 nm, between about 1 nm and about 25 nm, betweenabout 1 nm and about 15 nm, or between about 1 nm and about 8 nm. Thenanoparticle has a fluorescent compound positioned within thenanoparticle, and has greater brightness and fluorescent quantum yieldthan the free fluorescent compound. The nanoparticle also exhibits highbiostability and biocompatibility. To facilitate efficient urinaryexcretion of the nanoparticle, it may be coated with an organic polymer,such as poly(ethylene glycol) (PEG). The small size of the nanoparticle,the silica base and the organic polymer coating minimizes the toxicityof the nanoparticle when administered in vivo. In order to target aspecific cell type, the nanoparticle may further be conjugated to aligand, which is capable of binding to a cellular component (e.g., thecell membrane or other intracellular component) associated with thespecific cell type, such as a tumor marker or a signaling pathwayintermediate. In one embodiment, a therapeutic agent may be attached tothe nanoparticle. To permit the nanoparticle to be detectable by notonly optical imaging (such as fluorescence imaging), but also otherimaging techniques, such as positron emission tomography (PET), singlephoton emission computed tomography (SPECT), computerized tomography(CT), and magnetic resonance imaging (MRI), the nanoparticle may also beconjugated to a contrast agent, such as a radionuclide.

The properties of the nanoparticles enable excretion through thekidneys, as well as selective uptake and retention in tumors comparedwith normal tissues. This, along with the lack of in vivo toxicity, hasresulted in a unique product that is promising for translation to theclinic.

The nanoparticle may have both a ligand and a contrast agent. The ligandallows for the nanoparticle to target a specific cell type through thespecific binding between the ligand and the cellular component. Thistargeting, combined with multimodal imaging, has multiple uses. Forexample, the nanoparticles can be used to map sentinel lymph node (SLN),as well as to mark the tumor margins or neural structures, enabling thesurgeon to resect malignant lesions under direct visualization and toobviate complications during the surgical procedure. The ligand may alsofacilitate entry of the nanoparticle into the cell or barrier transport,for example, for assaying the intracellular environment.

The nanoparticle can be coupled with a ligand and a therapeutic agentwith or without a radiolabel. The radiolabel can additionally serve as atherapeutic agent for creating a multitherapeutic platform. Thiscoupling allows the therapeutic agent to be delivered to the specificcell type through the specific binding between the ligand and thecellular component. This specific targeting of the therapeutic agentensures selective treatment of the disease site with minimum sideeffects.

The fluorescent nanoparticle of the present invention includes asilica-based core comprising a fluorescent compound positioned withinthe core, and a silica shell on the core. The silica shell may surroundat least a portion of the core. Alternatively, the nanoparticle may haveonly the core and no shell. The core of the nanoparticle may contain thereaction product of a reactive fluorescent compound and a co-reactiveorgano-silane compound. In another embodiment, the core of thenanoparticle may contain the reaction product of a reactive fluorescentcompound and a co-reactive organo-silane compound, and silica. Thediameter of the core may be from about 0.05 nm to about 100 nm, fromabout 0.1 nm to about 50 nm, from about 0.5 nm to about 25 nm, fromabout 0.8 nm to about 15 nm, or from about 1 nm to about 8 nm. The shellof the nanoparticle can be the reaction product of a silica formingcompound. The shell of the nanoparticle may have a range of layers. Forexample, the silica shell may be from about 1 to about 20 layers, fromabout 1 to about 15 layers, from about 1 to about 10 layers, or fromabout 1 to about 5 layers. The thickness of the shell may range fromabout 0.01 nm to about 90 nm, from about 0.02 nm to about 40 nm, fromabout 0.05 nm to about 20 nm, from about 0.05 nm to about 10 nm, or fromabout 0.05 nm to about 5 nm.

The silica shell of the nanoparticle may cover only a portion ofnanoparticle or the entire particle. For example, the silica shell maycover about 1 to about 100 percent, from about 10 to about 80 percent,from about 20 to about 60 percent, or from about 30 to about 50 percentof the nanoparticle. The silica shell can be either solid, i.e.,substantially non-porous, meso-porous, such as semi-porous, or porous.

The present fluorescent nanoparticle may be synthesized by the steps of:covalently conjugating a fluorescent compound, such as a reactivefluorescent dye, with the reactive moieties including, but not limitedto, maleimide, iodoacetamide, thiosulfate, amine, N-Hydroxysuccimideester, 4-sulfo-2,3,5,6-tetrafluorophenyl (STP) ester, sulfosuccinimidylester, sulfodichlorophenol esters, sulfonyl chloride, hydroxyl,isothiocyanate, carboxyl, to an organo-silane compound, such as aco-reactive organo-silane compound, to form a fluorescent silicaprecursor, and reacting the fluorescent silica precursor to form afluorescent core; covalently conjugating a fluorescent compound, such asa reactive fluorescent dye, to an organo-silane compound, such as aco-reactive organo-silane compound, to form a fluorescent silicaprecursor, and reacting the fluorescent silica precursor with a silicaforming compound, such as tetraalkoxysilane, to form a fluorescent core;and reacting the resulting core with a silica forming compound, such asa tetraalkoxysilane, to form a silica shell on the core, to provide thefluorescent nanoparticle.

The synthesis of the fluorescent monodisperse core-shell nanoparticlesis based on a two-step process. First, the organic dye molecules,tetramethylrhodamine isothiocynate (TRITC), are covalently conjugated toa silica precursor and condensed to form a dye-rich core. Second, thesilica gel monomers are added to form a denser silica network around thefluorescent core material, providing shielding from solvent interactionsthat can be detrimental to photostability. The versatility of thepreparative route allows for the incorporation of different fluorescentcompounds, such as fluorescent organic compounds or dyes, depending onthe intended nanoparticle application. The fluorescent compounds thatmay be incorporated in the dye-rich core can cover the entire UV-Vis tonear-IR absorption and emission spectrum. U.S. patent application Ser.Nos. 10/306,614, 10/536,569 and 11/119,969. Wiesner et al., Peg-coatedCore-shell Silica Nanoparticles and Mathods of Manufactire and Use,PCT/US2008/74894.

For the synthesis of the compact core-shell nanoparticle, the dyeprecursor is added to a reaction vessel that contains appropriateamounts of ammonia, water and solvent and allowed to react overnight.The dye precursor is synthesized by addition reaction between a specificnear-infrared dye of interest and 3-aminopropyltriethoxysilane in molarratio of 1:50, in exclusion of moisture. After the synthesis of thedye-rich compact core is completed, tetraethylorthosilicate (TEOS) issubsequently added to grow the silica shell that surrounded the core.

The synthesis of the expanded core-shell nanoparticle is accomplished byco-condensing TEOS with the dye precursor and allowing the mixture toreact overnight. After the synthesis of the expanded core is completed,additional TEOS is added to grow the silica shell that surrounded thecore.

The synthesis of the homogenous nanoparticles is accomplished byco-condensing all the reagents, the dye precursor and TEOS and allowingthe mixture to react overnight.

The nanoparticles may incorporate any known fluorescent compound, suchas fluorescent organic compound, dyes, pigments, or combinationsthereof. A wide variety of suitable chemically reactive fluorescent dyesare known, see for example MOLECULAR PROBES HANDBOOK OF FLUORESCENTPROBES AND RESEARCH CHEMICALS, 6th ed., R. P. Haugland, ed. (1996). Atypical fluorophore is, for example, a fluorescent aromatic orheteroaromatic compound such as is a pyrene, an anthracene, anaphthalene, an acridine, a stilbene, an indole or benzindole, anoxazole or benzoxazole, a thiazole or benzothiazole, a4-amino-7-nitrobenz-2-oxa-1,3-diazole (NBD), a cyanine, a carbocyanine,a carbostyryl, a porphyrin, a salicylate, an anthranilate, an azulene, aperylene, a pyridine, a quinoline, a coumarin (includinghydroxycoumarins and aminocoumarins and fluorinated derivativesthereof), and like compounds, see for example U.S. Pat. Nos. 5,830,912,4,774,339, 5,187,288, 5,248,782, 5,274,113, 5,433,896, 4,810,636 and4,812,409. In one embodiment, Cy5, a near infrared fluorescent (NIRF)dye, is positioned within the silica core of the present nanoparticle.Near infrared-emitting probes exhibit decreased tissue attenuation andautofluorescence. Burns et al. “Fluorescent silica nanoparticles withefficient urinary excretion for nanomedicine”, Nano Letters, 2009, 9(1), 442-448.

Non-limiting fluorescent compound that may be used in the presentinvention include, Cy5, Cy5.5 (also known as Cy5++), Cy2, fluoresceinisothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC),phycoerythrin, Cy7, fluorescein (FAM), Cy3, Cy3.5 (also known as Cy3++),Texas Red, LightCycler-Red 640, LightCycler Red 705,tetramethylrhodamine (TMR), rhodamine, rhodamine derivative (ROX),hexachlorofluorescein (HEX), rhodamine 6G (R6G), the rhodaminederivative JA133, Alexa Fluorescent Dyes (such as Alexa Fluor 488, AlexaFluor 546, Alexa Fluor 633, Alexa Fluor 555, and Alexa Fluor 647),4′,6-diamidino-2-phenylindole (DAPI), Propidium iodide, AMCA, SpectrumGreen, Spectrum Orange, Spectrum Aqua, Lissamine, and fluorescenttransition metal complexes, such as europium. Fluorescent compound thatcan be used also include fluorescent proteins, such as GFP (greenfluorescent protein), enhanced GFP (EGFP), blue fluorescent protein andderivatives (BFP, EBFP, EBFP2, Azurite, mKalamal), cyan fluorescentprotein and derivatives (CFP, ECFP, Cerulean, CyPet) and yellowfluorescent protein and derivatives (YFP, Citrine, Venus, YPet).WO2008142571, WO2009056282, WO9922026.

The silica shell surface of the nanoparticles can be modified by usingknown cross-linking agents to introduce surface functional groups.Crosslinking agents include, but are not limited to, divinyl benzene,ethylene glycol dimethacrylate, trimethylol propane trimethacrylate,N,N′-methylene-bis-acrylamide, alkyl ethers, sugars, peptides, DNAfragments, or other known functionally equivalent agents. The ligand maybe conjugated to the nanoparticle of the present invention by, forexample, through coupling reactions using carbodiimide, carboxylates,esters, alcohols, carbamides, aldehydes, amines, sulfur oxides, nitrogenoxides, halides, or any other suitable compound known in the art. U.S.Pat. No. 6,268,222.

An organic polymer may be attached to the present nanoparticle, e.g.,attached to the surface of the nanoparticle. An organic polymer may beattached to the silica shell of the present nanoparticle. The organicpolymer that may be used in the present invention include PEG,polylactate, polylactic acids, sugars, lipids, polyglutamic acid (PGA),polyglycolic acid, poly(lactic-co-glycolic acid) (PLGA), polyvinylacetate (PVA), and the combinations thereof. The attachment of theorganic polymer to the nanoparticle may be accomplished by a covalentbond or non-covalent bond, such as by ionic bond, hydrogen bond,hydrophobic bond, coordination, adhesive, and physical absorption. Inone embodiment, the nanoparticle is covalently conjugated with PEG,which prevents adsorption of serum proteins, facilitates efficienturinary excretion and decreases aggregation of the nanoparticle. Burnset al. “Fluorescent silica nanoparticles with efficient urinaryexcretion for nanomedicine”, Nano Letters, 2009, 9 (1), 442-448.

The surface of the nanoparticle may be modified to incorporate at leastone functional group. The organic polymer (e.g., PEG) attached to thenanoparticle may be modified to incorporate at least one functionalgroup. For example, the functional group can be a maleimide orN-Hydroxysuccinimide (NHS) ester. The incorporation of the functionalgroup makes it possible to attach various ligands, contrast agentsand/or therapeutic agents to the nanoparticle.

A ligand may be attached to the present nanoparticle. The ligand iscapable of binding to at least one cellular component. The cellularcomponent may be associated with specific cell types or having elevatedlevels in specific cell types, such as cancer cells or cells specific toparticular tissues and organs. Accordingly, the nanoparticle can targeta specific cell type, and/or provides a targeted delivery for thetreatment and diagnosis of a disease. As used herein, the term “ligand”refers to a molecule or entity that can be used to identify, detect,target, monitor, or modify a physical state or condition, such as adisease state or condition. For example, a ligand may be used to detectthe presence or absence of a particular receptor, expression level of aparticular receptor, or metabolic levels of a particular receptor. Theligand can be, for example, a peptide, a protein, a protein fragment, apeptide hormone, a sugar (i.e., lectins), a biopolymer, a syntheticpolymer, an antigen, an antibody, an antibody fragment (e.g., Fab,nanobodies), an aptamer, a virus or viral component, a receptor, ahapten, an enzyme, a hormone, a chemical compound, a pathogen, amicroorganism or a component thereof, a toxin, a surface modifier, suchas a surfactant to alter the surface properties or histocompatability ofthe nanoparticle or of an analyte when a nanoparticle associatestherewith, and combinations thereof. Preferred ligands are, for example,antibodies, such as monoclonal or polyclonal antibodies, and receptorligands. In another embodiment, the ligand is poly-L-lysine (pLysine).

An antigen may be attached to the nanoparticle. The antigen-attachednanoparticle may be used for vaccination.

The terms “component of a cell” or “cellular component” refer to, forexample, a receptor, an antibody, a hapten, an enzyme, a hormone, abiopolymer, an antigen, a nucleic acid (DNA or RNA), a microorganism, avirus, a pathogen, a toxin, combinations thereof, and like components.The component of a cell may be positioned on the cell (e.g., atransmembrane receptor) or inside the cell. In one embodiment, thecomponent of a cell is a tumor marker. As used herein, the term “tumormarker” refers to a molecule, entity or substance that is expressed oroverexpressed in a cancer cell but not normal cell. For example, theoverexpression of certain receptors is associated with many types ofcancer. A ligand capable of binding to a tumor marker may be conjugatedto the surface of the present nanoparticle, so that the nanoparticle canspecifically target the tumor cell.

A ligand may be attached to the present nanoparticle directly or througha linker. The attachment of the ligand to the nanoparticle may beaccomplished by a covalent bond or non-covalent bond, such as by ionicbond, hydrogen bond, hydrophobic bond, coordination, adhesive, andphysical absorption. The ligand may be coated onto the surface of thenanoparticle. The ligand may be imbibed into the surface of thenanoparticle. As used herein, “imbibe” refers to assimilation or takingin. The ligand may be attached to the surface of the fluorescentnanoparticle, or may be attached to the core when the shell is porous oris covering a portion of the core. When the ligand is attached to thenanoparticle through a linker, the linker can be any suitable molecules,such as a functionalized PEG. The PEGs can have multiple functionalgroups for attachment to the nanoparticle and ligands. The particle canhave different types of functionalized PEGs bearing different functionalgroups that can be attached to multiple ligands. This can enhancemultivalency effects and/or contrast at the target site, which allowsthe design and optimization of a complex multimodal platform withimproved targeted detection, treatment, and sensing in vivo.

A variety of different ligands may be attached to the nanoparticle. Forexample, tripeptide Arg-Gly-Asp (RGD) may be attached to thenanoparticle. Alternatively, cyclic peptide cRGD (which may containother amino acid(s), e.g., cRGDY) may be attached to the nanoparticle.Any linear, cyclic or branched peptide containing the RGD sequence iswithin the scope of the present invention. RGD binds to α_(v)β₃integrin, which is overexpressed at the surface of activated endothelialcells during angiogenesis and in various types of tumor cells.Expression levels of α_(v)β₃ integrin have been shown to correlate wellwith the aggressiveness of tumors. Ruoslahti et al. New perspectives incell adhesion: RGD and integrins. Science 1987; 238:491. Gladson et al.Glioblastoma expression of vitronectin and alpha v beta 3 integrin.Adhesion mechanism for transformed glial cells. J. Clin. Invest. 1991;88:1924-1932. Seftor et al. Role of the alpha v beta 3 integrin in humanmelanoma cell invasion. Proc. Natl. Acad. Sci. 1992; 89:1557-1561.

Alternatively, synthetic peptide EPPT1 may be the ligand attached to thenanoparticle. EPPT1, derived from the monoclonal antibody (ASM2) bindingsite, targets underglycosylated MUC1 (uMUC1). MUC1, a transmembranereceptor, is heavily glycosylated in normal tissues; however, it isoverexpressed and aberrantly underglycosylated in almost all humanepithelial cell adenocarcinomas, and is implicated in tumorpathogenesis. Moore et al. In vivo targeting of underglycosylated MUC-1tumor antigen using a multimodal imaging probe. Cancer Res. 2004;64:1821-7. Patel et al. MUC1 plays a role in tumor maintenance inaggressive thryroid carcinomas. Surgery. 2005; 138:994-1001. Specificantibodies including monoclonal antibodies against uMUC1 mayalternatively be conjugated to the nanoparticle in order to targetuMUC1.

In one embodiment, peptide analogues of α-melanotropin stimulatinghormone (α-MSH) are the ligands attached to the nanoparticle. Peptideanalogues of α-MSH are capable of binding to melanocortin-1 receptors(MC1R), a family of G-protein-coupled receptors overexpressed inmelanoma cells. Loir et al. Cell Mol. Biol. (Noisy-le-grand) 1999,45:1083-1092.

In another embodiment, octreotate, a peptide analog of 14-amino acidsomatostatin, is the ligand attached to the nanoparticle. Octreotide,which has a longer half-life than somatostatin, is capable of binding tosomatostatin receptor (SSTR). SSTR, a member of the G-protein coupledreceptor family, is overexpressed on the surface of several humantumors. Reubi et al. Distribution of Somatostatin Receptors in Normaland Tumor-Tissue. Metab. Clin. Exp. 1990; 39:78-81. Reubi et al.Somatostatin receptors and their subtypes in human tumors and inperitumoral vessels. Metab. Clin. Exp. 1996; 45:39-41. Othersomatostatin analogs may alternatively be conjugated to the nanoparticleto target SSTR, such as Tyr3-octreotide (Y3-OC), octreotate (TATE),Tyr3-octreotate (Y3-TATE), and ¹¹¹In-DTPA-OC. These somatostatinanalogues may be utilized for both PET diagnostic imaging and targetedradiotherapy of cancer. de Jong et al. Internalization of radiolabelled[DTPA⁰]octreotide and [DOTA⁰, Tyr³]octreotide: peptides for somatostatinreceptor targeted scintigraphy and radionuclide therapy. Nucl. Med.Commun. 1998; 19:283-8. de Jong et al. Comparison of ¹¹¹In-LabeledSomatostatin Analogues for Tumor Scintigraphy and Radionuclide Therapy.Cancer Res. 1998; 58:437-41. Lewis et al. Comparison of four⁶⁴Cu-labeled somatostatin analogs in vitro and in a tumor-bearing ratmodel: evaluation of new derivatives for PET imaging and targetedradiotherapy. J Med Chem 1999; 42:1341-7. Krenning et al. SomatostatinReceptor Scintigraphy with Indium-111-DTPA-D-Phe-1-Octreotide in Man:Metabolism, Dosimetry and Comparison with Iodine-123-Tyr-3-Octreotide. JNucl. Med. 1992; 33:652-8.

The number of ligands attached to the nanoparticle may range from about1 to about 20, from about 2 to about 15, from about 3 to about 10, fromabout 1 to about 10, or from about 1 to about 6. The small number of theligands attached to the nanoparticle helps maintain the hydrodynamicdiameter of the present nanoparticle which meet the renal clearancecutoff size range. Hilderbrand et al., Near-infrared fluorescence:application to in vivo molecular imaging, Curr. Opin. Chem. Biol.,14:71-9, 2010. The number of ligands measured may be an average numberof ligands attached to more than one nanoparticle. Alternatively, onenanoparticle may be measured to determine the number of ligandsattached. The number of ligands attached to the nanoparticle can bemeasured by any suitable methods, which may or may not be related to theproperties of the ligands. For example, the number of cRGD peptidesbound to the particle may be estimated using FCS-based measurements ofabsolute particle concentrations and the starting concentration of thereagents for cRGD peptide. Average number of RGD peptides pernanoparticle and coupling efficiency of RGD to functionalized PEG groupscan be assessed colorimetrically under alkaline conditions and Biuretspectrophotometric methods. The number of ligands attached to thenanoparticle may also be measured by nuclear magnetic resonance (NMR),optical imaging, assaying radioactivity, etc. The method can be readilydetermined by those of skill in the art.

A contrast agent may be attached to the present nanoparticle for medicalor biological imaging. As used herein, the term “contrast agent” refersto a substance, molecule or compound used to enhance the visibility ofstructures or fluids in medical or biological imaging. The term“contrast agent” also refers to a contrast-producing molecule. Theimaging techniques encompassed by the present invention include positronemission tomography (PET), single photon emission computed tomography(SPECT), computerized tomography (CT), magnetic resonance imaging (MRI),optical bioluminescence imaging, optical fluorescence imaging, andcombinations thereof. The contrast agent encompassed by the presentinvention may be any molecule, substance or compound known in the artfor PET, SPECT, CT, MRI, and optical imaging. The contrast agent may beradionuclides, radiometals, positron emitters, beta emitters, gammaemitters, alpha emitters, paramagnetic metal ions, and supraparamagneticmetal ions. The contrast agents include, but are not limited to, iodine,fluorine, copper, zirconium, lutetium, astatine, yttrium, gallium,indium, technetium, gadolinium, dysprosium, iron, manganese, barium andbarium sulfate. The radionuclides that may be used as the contrast agentattached to the nanoparticle of the present invention include, but arenot limited to, ⁸⁹Zr, ⁶⁴Cu, ⁶⁸Ga, ⁸⁶Y, ¹²⁴I and ¹⁷⁷Lu.

The contrast agent may be directly conjugated to the nanoparticle.Alternatively, the contrast agent may be indirectly conjugated to thenanoparticle, by attaching to linkers or chelates. The chelate may beadapted to bind a radionuclide. The chelates that can be attached to thepresent nanoparticle may include, but are not limited to,1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA),diethylenetriaminepentaacetic (DTPA), desferrioxamine (DFO) andtriethylenetetramine (TETA).

Suitable means for imaging, detecting, recording or measuring thepresent nanoparticles may also include, for example, a flow cytometer, alaser scanning cytometer, a fluorescence micro-plate reader, afluorescence microscope, a confocal microscope, a bright-fieldmicroscope, a high content scanning system, and like devices. More thanone imaging techniques may be used at the same time or consecutively todetect the present nanoparticles. In one embodiment, optical imaging isused as a sensitive, high-throughput screening tool to acquire multipletime points in the same subject, permitting semi-quantitativeevaluations of tumor marker levels. This offsets the relativelydecreased temporal resolution obtained with PET, although PET is neededto achieve adequate depth penetration for acquiring volumetric data, andto detect, quantitate, and monitor changes in receptor and/or othercellular marker levels as a means of assessing disease progression orimprovement, as well as stratifying patients to suitable treatmentprotocols.

A therapeutic agent may be attached to the fluorescent nanoparticle, forexample, for targeted treatment of a disease. The therapeutic agent maybe delivered to a diseased site in a highly specific or localized mannerwith release of the therapeutic agent in the disease site.Alternatively, the therapeutic agent may not be released. Thefluorescent nanoparticle conjugated with the ligand can be used fortargeted delivery of a therapeutic agent to a desired location in avariety of systems, such as on, or within, a cell or cell component,within the body of an organism, such as a human, or across theblood-brain barrier.

The therapeutic agent may be attached to the nanoparticle directly orindirectly. The therapeutic agent can be absorbed into the intersticesor pores of the silica shell, or coated onto the silica shell of thefluorescent nanoparticle. In other embodiments where the silica shell isnot covering all of the surface, the therapeutic agent can be associatedwith the fluorescent core, such as by physical absorption or by bondinginteraction. The therapeutic agent may be associated with the ligandthat is attached to the fluorescent nanoparticle. The therapeutic agentmay also be associated with the organic polymer or the contrast agent.For example, the therapeutic agent may be attached to the nanoparticlethrough PEG. The PEGs can have multiple functional groups for attachmentto the nanoparticle and therapeutic agent. The particle can havedifferent types of functionalized PEGs bearing different functionalgroups that can be attached to multiple therapeutic agents. Thetherapeutic agent may be attached to the nanoparticle covalently ornon-covalently.

As used herein, the term “therapeutic agent” refers to a substance thatmay be used in the diagnosis, cure, mitigation, treatment, or preventionof disease in a human or another animal. Such therapeutic agents includesubstances recognized in the official United States Pharmacopeia,official Homeopathic Pharmacopeia of the United States, officialNational Formulary, or any supplement thereof.

Therapeutic agents that can be incorporated with the fluorescentnanoparticles or the ligated-fluorescent nanoparticles of the inventioninclude nucleosides, nucleoside analogs, small interfering RNA (siRNA),microRNA, oligopeptides, polypeptides, antibodies, COX-2 inhibitors,apoptosis promoters, urinary tract agents, vaginal agents, vasodilatorsneurodegenerative agents (e.g., Parkinson's disease), obesity agents,ophthalmic agents, osteoporosis agents, para-sympatholytics,para-sympathometics, antianesthetics, prostaglandins, psychotherapeuticagents, respiratory agents, sedatives, hypnotics, skin and mucousmembrane agents, anti-bacterials, anti-fungals, antineoplastics,cardioprotective agents, cardiovascular agents, anti-thrombotics,central nervous system stimulants, cholinesterase inhibitors,contraceptives, dopamine receptor agonists, erectile dysfunction agents,fertility agents, gastrointestinal agents, gout agents, hormones,immunomodulators, suitably functionalized analgesics or general or localanesthetics, anti-convulsants, anti-diabetic agents, anti-fibroticagents, anti-infectives, motion sickness agents, muscle relaxants,immuno-suppressive agents, migraine agents, non-steroidalanti-inflammatory drugs (NSAIDs), smoking cessation agents, orsympatholytics (see Physicians' Desk Reference, 55th ed., 2001, MedicalEconomics Company, Inc., Montvale, N.J., pages 201-202).

Therapeutic agents that may be attached to the present nanoparticleinclude, but are not limited to, DNA alkylating agents, topoisomeraseinhibitors, endoplasmic reticulum stress inducing agents, a platinumcompound, an antimetabolite, vincalkaloids, taxanes, epothilones, enzymeinhibitors, receptor antagonists, therapeutic antibodies, tyrosinekinase inhibitors, boron radiosensitizers (i.e. velcade), andchemotherapeutic combination therapies.

Non-limiting examples of DNA alkylating agents are nitrogen mustards,such as Mechlorethamine, Cyclophosphamide (Ifosfamide, Trofosfamide),Chlorambucil (Melphalan, Prednimustine), Bendamustine, Uramustine andEstramustine; nitrosoureas, such as Carmustine (BCNU), Lomustine(Semustine), Fotemustine, Nimustine, Ranimustine and Streptozocin; alkylsulfonates, such as Busulfan (Mannosulfan, Treosulfan); Aziridines, suchas Carboquone, ThioTEPA, Triaziquone, Triethylenemelamine; Hydrazines(Procarbazine); Triazenes such as Dacarbazine and Temozolomide;Altretamine and Mitobronitol.

Non-limiting examples of Topoisomerase I inhibitors include Campothecinderivatives including CPT-11 (irinotecan), SN-38, APC, NPC, campothecin,topotecan, exatecan mesylate, 9-nitrocamptothecin, 9-aminocamptothecin,lurtotecan, rubitecan, silatecan, gimatecan, diflomotecan, extatecan,BN-80927, DX-8951f, and MAG-CPT as described in Pommier Y. (2006) Nat.Rev. Cancer 6(10):789-802 and U.S. Patent Publication No. 200510250854;Protoberberine alkaloids and derivatives thereof including berberrubineand coralyne as described in Li et al. (2000) Biochemistry39(24):7107-7116 and Gatto et al. (1996) Cancer Res. 15(12):2795-2800;Phenanthroline derivatives including Benzo[i]phenanthridine, Nitidine,and fagaronine as described in Makhey et al. (2003) Bioorg. Med. Chem.11 (8): 1809-1820; Terbenzimidazole and derivatives thereof as describedin Xu (1998) Biochemistry 37(10):3558-3566; and Anthracyclinederivatives including Doxorubicin, Daunorubicin, and Mitoxantrone asdescribed in Foglesong et al. (1992) Cancer Chemother. Pharmacol.30(2):123-]25, Crow et al. (1994) J. Med. Chem. 37(19):31913194, andCrespi et al. (1986) Biochem. Biophys. Res. Commun. 136(2):521-8.Topoisomerase II inhibitors include, but are not limited to Etoposideand Teniposide. Dual topoisomerase I and II inhibitors include, but arenot limited to, Saintopin and other Naphthecenediones, DACA and otherAcridine-4-Carboxamindes, Intoplicine and other Benzopyridoindoles,TAS-I03 and other 7H-indeno[2,1-c]Quinoline-7-ones, Pyrazoloacridine, XR11576 and other Benzophenazines, XR 5944 and other Dimeric compounds,7-oxo-7H-dibenz[f,ij]Isoquinolines and 7-oxo-7H-benzo[e]Perimidines, andAnthracenyl-amino Acid Conjugates as described in Denny and Baguley(2003) Curr. Top. Med. Chem. 3(3):339-353. Some agents inhibitTopoisomerase II and have DNA intercalation activity such as, but notlimited to, Anthracyclines (Aclarubicin, Daunorubicin, Doxorubicin,Epirubicin, Idarubicin, Amrubicin, Pirarubicin, Valrubicin, Zorubicin)and Antracenediones (Mitoxantrone and Pixantrone).

Examples of endoplasmic reticulum stress inducing agents include, butare not limited to, dimethyl-celecoxib (DMC), nelfinavir, celecoxib, andboron radiosensitizers (i.e. velcade (Bortezomib)).

Non-limiting examples of platinum based compound include Carboplatin,Cisplatin, Nedaplatin, Oxaliplatin, Triplatin tetranitrate, Satraplatin,Aroplatin, Lobaplatin, and JM-216. (see McKeage et al. (1997) J. Clin.Oncol. 201:1232-1237 and in general, CHEMOTHERAPY FOR GYNECOLOGICALNEOPLASM, CURRENT THERAPY AND NOVEL APPROACHES, in the Series Basic andClinical Oncology, Angioli et al. Eds., 2004).

Non-limiting examples of antimetabolite agents include Folic acid based,i.e. dihydrofolate reductase inhibitors, such as Aminopterin,Methotrexate and Pemetrexed; thymidylate synthase inhibitors, such asRaltitrexed, Pemetrexed; Purine based, i.e. an adenosine deaminaseinhibitor, such as Pentostatin, a thiopurine, such as Thioguanine andMercaptopurine, a halogenated/ribonucleotide reductase inhibitor, suchas Cladribine, Clofarabine, Fludarabine, or a guanine/guanosine:thiopurine, such as Thioguanine; or Pyrimidine based, i.e.cytosine/cytidine: hypomethylating agent, such as Azacitidine andDecitabine, a DNA polymerase inhibitor, such as Cytarabine, aribonucleotide reductase inhibitor, such as Gemcitabine, or athymine/thymidine: thymidylate synthase inhibitor, such as aFluorouracil (5-FU). Equivalents to 5-FU include prodrugs, analogs andderivative thereof such as 5′-deoxy-5-fluorouridine (doxifluroidine),1-tetrahydrofuranyl-5-fluorouracil (ftorafur), Capecitabine (Xeloda),S-I (MBMS-247616, consisting of tegafur and two modulators, a5-chloro-2,4dihydroxypyridine and potassium oxonate), ralititrexed(tomudex), nolatrexed (Thymitaq, AG337), LY231514 and ZD9331, asdescribed for example in Papamicheal (1999) The Oncologist 4:478-487.

Examples of vincalkaloids, include, but are not limited to Vinblastine,Vincristine, Vinflunine, Vindesine and Vinorelbine.

Examples of taxanes include, but are not limited to docetaxel,Larotaxel, Ortataxel, Paclitaxel and Tesetaxel. An example of anepothilone is iabepilone.

Examples of enzyme inhibitors include, but are not limited tofarnesyltransferase inhibitors (Tipifamib); CDK inhibitor (Alvocidib,Seliciclib); proteasome inhibitor (Bortezomib); phosphodiesteraseinhibitor (Anagrelide; rolipram); IMP dehydrogenase inhibitor(Tiazofurine); and lipoxygenase inhibitor (Masoprocol). Examples ofreceptor antagonists include, but are not limited to ERA (Atrasentan);retinoid X receptor (Bexarotene); and a sex steroid (Testolactone).

Examples of therapeutic antibodies include, but are not limited toanti-HER1/EGFR (Cetuximab, Panitumumab); Anti-HER2/neu (erbB2) receptor(Trastuzumab); Anti-EpCAM (Catumaxomab, Edrecolomab) Anti-VEGF-A(Bevacizumab); Anti-CD20 (Rituximab, Tositumomab, Ibritumomab);Anti-CD52 (Alemtuzumab); and Anti-CD33 (Gemtuzumab). U.S. Pat. Nos.5,776,427 and 7,601,355.

Examples of tyrosine kinase inhibitors include, but are not limited toinhibitors to ErbB: HER1/EGFR (Erlotinib, Gefitinib, Lapatinib,Vandetanib, Sunitinib, Neratinib); HER2/neu (Lapatinib, Neratinib); RTKclass III: C-kit (Axitinib, Sunitinib, Sorafenib), FLT3 (Lestaurtinib),PDGFR (Axitinib, Sunitinib, Sorafenib); and VEGFR (Vandetanib,Semaxanib, Cediranib, Axitinib, Sorafenib); bcr-abl (Imatinib,Nilotinib, Dasatinib); Src (Bosutinib) and Janus kinase 2(Lestaurtinib).

Chemotherapeutic agents that can be attached to the present nanoparticlemay also include amsacrine, Trabectedin, retinoids (Alitretinoin,Tretinoin), Arsenic trioxide, asparagine depleterAsparaginase/Pegaspargase), Celecoxib, Demecolcine, Elesclomol,Elsamitrucin, Etoglucid, Lonidamine, Lucanthone, Mitoguazone, Mitotane,Oblimersen, Temsirolimus, and Vorinostat.

Examples of specific therapeutic agents that can be linked, ligated, orassociated with the fluorescent nanoparticles of the invention areflomoxef; fortimicin(s); gentamicin(s); glucosulfone solasulfone;gramicidin S; gramicidin(s); grepafloxacin; guamecycline; hetacillin;isepamicin; josamycin; kanamycin(s); flomoxef; fortimicin(s);gentamicin(s); glucosulfone solasulfone; gramicidin S; gramicidin(s);grepafloxacin; guamecycline; hetacillin; isepamicin; josamycin;kanamycin(s); bacitracin; bambermycin(s); biapenem; brodimoprim;butirosin; capreomycin; carbenicillin; carbomycin; carumonam;cefadroxil; cefamandole; cefatrizine; cefbuperazone; cefclidin;cefdinir; cefditoren; cefepime; cefetamet; cefixime; cefinenoxime;cefininox; cladribine; apalcillin; apicycline; apramycin; arbekacin;aspoxicillin; azidamfenicol; aztreonam; cefodizime; cefonicid;cefoperazone; ceforamide; cefotaxime; cefotetan; cefotiam; cefozopran;cefpimizole; cefpiramide; cefpirome; cefprozil; cefroxadine; cefteram;ceftibuten; cefuzonam; cephalexin; cephaloglycin; cephalosporin C;cephradine; chloramphenicol; chlortetracycline; clinafloxacin;clindamycin; clomocycline; colistin; cyclacillin; dapsone;demeclocycline; diathymosulfone; dibekacin; dihydrostreptomycin;6-mercaptopurine; thioguanine; capecitabine; docetaxel; etoposide;gemcitabine; topotecan; vinorelbine; vincristine; vinblastine;teniposide; melphalan; methotrexate; 2-p-sulfanilyanilinoethanol;4,4′-sulfinyldianiline; 4-sulfanilamidosalicylic acid; butorphanol;nalbuphine. streptozocin; doxorubicin; daunorubicin; plicamycin;idarubicin; mitomycin C; pentostatin; mitoxantrone; cytarabine;fludarabine phosphate; butorphanol; nalbuphine. streptozocin;doxorubicin; daunorubicin; plicamycin; idarubicin; mitomycin C;pentostatin; mitoxantrone; cytarabine; fludarabine phosphate;acediasulfone; acetosulfone; amikacin; amphotericin B; ampicillin;atorvastatin; enalapril; ranitidine; ciprofloxacin; pravastatin;clarithromycin; cyclosporin; famotidine; leuprolide; acyclovir;paclitaxel; azithromycin; lamivudine; budesonide; albuterol; indinavir;metformin; alendronate; nizatidine; zidovudine; carboplatin; metoprolol;amoxicillin; diclofenac; lisinopril; ceftriaxone; captopril; salmeterol;xinafoate; imipenem; cilastatin; benazepril; cefaclor; ceftazidime;morphine; dopamine; bialamicol; fluvastatin; phenamidine; podophyllinicacid 2-ethylhydrazine; acriflavine; chloroazodin; arsphenamine;amicarbilide; aminoquinuride; quinapril; oxymorphone; buprenorphine;floxuridine; dirithromycin; doxycycline; enoxacin; enviomycin;epicillin; erythromycin; leucomycin(s); lincomycin; lomefloxacin;lucensomycin; lymecycline; meclocycline; meropenem; methacycline;micronomicin; midecamycin(s); minocycline; moxalactam; mupirocin;nadifloxacin; natamycin; neomycin; netilmicin; norfloxacin;oleandomycin; oxytetracycline; p-sulfanilylbenzylamine; panipenem;paromomycin; pazufloxacin; penicillin N; pipacycline; pipemidic acid;polymyxin; primycin; quinacillin; ribostamycin; rifamide; rifampin;rifamycin SV; rifapentine; rifaximin; ristocetin; ritipenem;rokitamycin; rolitetracycline; rosaramycin; roxithromycin;salazosulfadimidine; sancycline; sisomicin; sparfloxacin; spectinomycin;spiramycin; streptomycin; succisulfone; sulfachrysoidine; sulfaloxicacid; sulfamidochrysoidine; sulfanilic acid; sulfoxone; teicoplanin;temafloxacin; temocillin; tetroxoprim; thiamphenicol; thiazolsulfone;thiostrepton; ticarcillin; tigemonam; tobramycin; tosufloxacin;trimethoprim; trospectomycin; trovafloxacin; tuberactinomycin;vancomycin; azaserine; candicidin(s); chlorphenesin; dermostatin(s);filipin; fungichromin; mepartricin; nystatin; oligomycin(s); perimycinA; tubercidin; 6-azauridine; 6-diazo-5-oxo-L-norleucine;aclacinomycin(s); ancitabine; anthramycin; azacitadine; azaserine;bleomycin(s); ethyl biscoumacetate; ethylidene dicoumarol; iloprost;lamifiban; taprostene; tioclomarol; tirofiban; amiprilose; bucillamine;gusperimus; gentisic acid; glucamethacin; glycol salicylate;meclofenamic acid; mefenamic acid; mesalamine; niflumic acid;olsalazine; oxaceprol; S-enosylmethionine; salicylic acid; salsalate;sulfasalazine; tolfenamic acid; carubicin; carzinophillin A;chlorozotocin; chromomycin(s); denopterin; doxifluridine; edatrexate;eflornithine; elliptinium; enocitabine; epirubicin; mannomustine;menogaril; mitobronitol; mitolactol; mopidamol; mycophenolic acid;nogalamycin; olivomycin(s); peplomycin; pirarubicin; piritrexim;prednimustine; procarbazine; pteropterin; puromycin; ranimustine;streptonigrin; thiamiprine; mycophenolic acid; procodazole; romurtide;sirolimus (rapamycin); tacrolimus; butethamine; fenalcomine;hydroxytetracaine; naepaine; orthocaine; piridocaine; salicyl alcohol;3-amino-4-hydroxybutyric acid; aceclofenac; alminoprofen; amfenac;bromfenac; bromosaligenin; bumadizon; carprofen; diclofenac; diflunisal;ditazol; enfenamic acid; etodolac; etofenamate; fendosal; fepradinol;flufenamic acid; Tomudex®(N-[[5-[[1,4-Dihydro-2-methyl-4-oxo-6-quinazolinyl)methyl]methylamino]-2-thienyl]carbonyl]-L-glutamicacid), trimetrexate, tubercidin, ubenimex, vindesine, zorubicin;argatroban; coumetarol or dicoumarol.

Lists of additional therapeutic agents can be found, for example, in:Physicians' Desk Reference, 55th ed., 2001, Medical Economics Company,Inc., Montvale, N.J.; USPN Dictionary of USAN and International DrugNames, 2000, The United States Pharmacopeial Convention, Inc.,Rockville, Md.; and The Merck Index, 12th ed., 1996, Merck & Co., Inc.,Whitehouse Station, N.J.

The therapeutic agent may also include radionuclides when the presentnanoparticle is used in targeted radiotherapy. In one embodiment, lowenergy beta-emitting radionuclides, such as ¹⁷⁷Lu-chelated constructs,is associated with the nanoparticle and used to treat relatively smalltumor burdens or micrometastatic disease. In another embodiment, higherenergy beta emitters, such as yttrium-90 (⁹⁰Y), may be used to treatlarger tumor burdens. Iodine-131 (¹³¹I) may also be used forradiotherapy.

The surface of the nanoparticle may be modified to incorporate at leastone functional group. The organic polymer (e.g., PEG) attached to thenanoparticle may be modified to incorporate at least one functionalgroup. For example, the functional group can be a maleimide orN-Hydroxysuccinimide (NHS) ester. The incorporation of the functionalgroup makes it possible to attach various ligands, contrast agentsand/or therapeutic agents to the nanoparticle.

In one embodiment, a therapeutic agent is attached to the nanoparticle(surface or the organic polymer coating) via an NHS ester functionalgroup. For example, tyrosine kinase inhibitor such as dasatinib (BMS) orchemotherapeutic agent (e.g., taxol), can be coupled via an ester bondto the nanoparticle. This ester bond can then be cleaved in an acidicenvironment or enzymatically in vivo. This approach may be used todeliver a prodrug to a subject where the drug is released from theparticle in vivo.

We have tested the prodrug approach by coupling small molecule inhibitordasatinib with the PEG molecules of the nanoparticle. Based onbiodistribution results and the human drug dosing calculations, thenanoparticle has been found to have unique biological properties,including relatively rapid clearance from the blood compared to tumorsand subsequent tumor tissue accumulation of the therapeutic agent, whichsuggests that a prodrug approach is feasible. The functionalizednanoparticle permits drugs to be dosed multiple times, ensuring that thedrug concentration in the tumor is greater than that specified by theIC-50 in tumor tissue, yet will not be dose-limiting to other organtissues, such as the heart, liver or kidney. The therapeutic agent andnanoparticle can be radiolabeled or optically labelled separately,allowing independent monitoring of the therapeutic agent and thenanoparticle. In one embodiment, radiofluorinated (i.e., ¹⁸F) dasatinibis coupled with PEG-3400 moieties attached to the nanoparticle via NHSester linkages. Radiofluorine is crucial for being able to independentlymonitor time-dependent changes in the distribution and release of thedrug from the radioiodinated (¹²⁴I) fluorescent (Cy5) nanoparticle. Inthis way, we can separately monitor the prodrug (dasatinib) andnanoparticle. This permits optimization of the prodrug design comparedwith methods in the prior art where no dual-labeling approach is used.In another embodiment, radiotherapeutic iodine molecules (i.e., I-131),or other therapeutic gamma or alpha emitters, are conjugated with PEGvia a maleimide functional group, where the therapeutic agent may notdissociate from the PEG in vivo.

In order for the present nanoparticle to readily accommodate largeranges of ligands, contrast agents or chelates, the surface of thenanoparticle may be modified to incorporate a functional group. Thenanoparticle may also be modified with organic polymers (e.g., PEGs) orchelates that can incorporate a functional group. In the meantime, theligand, contrast agent, or therapeutic agent is modified to incorporatea functional group that is able to react with the functional group onthe nanoparticle, or on the PEGs or chelating agents attached to thenanoparticle under suitable conditions. Accordingly, any ligand,contrast agent or therapeutic agent that has the reactive functionalgroup is able to be readily conjugated to the nanoparticle. Thisgeneralizable approach is referred herein as “click chemistry”, whichwould allow for a great deal of versatility to explore multimodalityapplications. Any suitable reaction mechanism may be adapted in thepresent invention for “click chemistry”, so long as facile andcontrolled attachment of the ligand, contrast agent or chelate to thenanoparticle can be achieved. In one embodiment, a free triple bond isintroduced onto PEG, which is already covalently conjugated with theshell of the nanoparticle. In the meantime, an azide bond is introducedonto the desired ligand (or contrast agent, chelate). When the PEGylatednanoparticle and the ligand (or contrast agent, chelate) are mixed inthe presence of a copper catalyst, cycloaddition of azide to the triplebond will occur, resulting in the conjugation of the ligand with thenanoparticle. In a second embodiment, a maleimide functional group and athiol group may be introduced onto the nanoparticle and the desiredligand (or contrast agent, chelate), with the nanoparticle having themaleimide functional group, the ligand (or contrast agent, chelate)having the thiol group, or vice versa. The double bond of maleimidereadily reacts with the thiol group to form a stable carbon-sulfur bond.In a third embodiment, an activated ester functional group, e.g., asuccinimidyl ester group, and an amine group may be introduced onto thenanoparticle and the desired ligand, contrast agent or chelate. Theactivated ester group readily reacts with the amine group to form astable carbon-nitrogen amide bond.

After administration of the present nanoparticle to a subject, the bloodresidence half-time of the nanoparticles may range from about 2 hours toabout 25 hours, from about 3 hours to about 20 hours, from about 3 hoursto about 15 hours, from about 4 hours to about 10 hours, or from about 5hours to about 6 hours. Longer blood residence half-time means longercirculation, which allows more nanoparticles to accumulate at the targetsite in vivo. Blood residence half-time may be evaluated as follows. Thenanoparticles are first administered to a subject (e.g., a mouse, aminiswine or a human). At various time points post administration, bloodsamples are taken to measure nanoparticle concentrations throughsuitable methods.

After administration of the present nanoparticle to a subject, the tumorresidence half-time of the present nanoparticles may range from about 5hours to about 5 days, from about 10 hours to about 4 days, from about15 hours to about 3.5 days, from about 20 hours to about 3 days, fromabout 2.5 days to about 3.1 days, from about 1 day to 3 days, or about73.5 hours.

The ratio of the tumor residence half-time to the blood residencehalf-time of the nanoparticle may range from about 2 to about 30, fromabout 3 to about 20, from about 4 to about 15, from about 4 to about 10,from about 10 to about 15, or about 13.

In one embodiment, to estimate residence (or clearance) half-time valuesof the radiolabeled nanoparticles (T_(1/2)) in blood, tumor, and othermajor organs/tissues, the percentage of the injected dose per gram (%ID/g) values are measured by sacrificing groups of mice at specifiedtimes following administration of the nanoparticles. Blood, tumor, andorgans are harvested, weighed, and counted in a scintillation γ-counter.The % ID/g values are corrected for radioactive decay to the time ofinjection. The resulting time-activity concentration data for eachtissue are fit to a decreasing monoexponential function to estimatetissue/organ T_(1/2) values.

After administration of the present nanoparticle to a subject, the renalclearance of the present nanoparticles may range from about 10% ID(initial dose) to about 100% ID in about 24 hours, from about 20% ID toabout 90% ID in about 24 hours, from about 30% ID to about 80% ID inabout 24 hours, from about 40% ID to about 70% ID in about 24 hours,from about 40% ID to about 60% ID in about 24 hours, from about 40% IDto about 50% ID in about 24 hours, or about 43% ID in about 24 hours.Renal clearance may be evaluated as follows. The nanoparticles are firstadministered to a subject (e.g., a mouse, a miniswine or a human). Atvarious time points post administration, urine samples are taken tomeasure nanoparticle concentrations through suitable methods.

In one embodiment, renal clearance (e.g., the fraction of nanoparticlesexcreted in the urine over time) is assayed as follows. A subject isadministered with the present nanoparticles, and urine samples collectedover a certain time period (e.g., 168 hours). Particle concentrations ateach time point are determined using fluorometric analyses and a serialdilution calibration curve generated from background-correctedfluorescence signal measurements of urine samples mixed with knownparticle concentrations (% ID). Concentration values, along withestimates of average daily mouse urine volumes, are used to computecumulative % ID/g urine excreted. In another embodiment, renal clearanceof radiolabeled nanoparticles is assayed by measuring urine specimenactivities (counts per minute) over similar time intervals using, forexample, γ-counting, and after nanoparticle administration to computecumulative urine excretion.

In a third embodiment, to assess cumulative fecal excretion, feces arecollected in metabolic cages over similar time intervals afteradministration of the nanoparticles and specimen activities determinedusing a γ-counter.

When the nanoparticles in the amount of about 100 times of the humandose equivalent are administered to a subject, substantially no anemia,weight loss, agitation, increased respiration, GI disturbance, abnormalbehavior, neurological dysfunction, abnormalities in hematology,abnormalities in clinical chemistries, drug-related lesions in organpathology, mortality, or combination thereof are observed in about 10 toabout 14 days.

When the present nanoparticle contains at least one attached ligand, themultivalency enhancement of the nanoparticle (e.g., compared to theligand alone) may range from about 1.5 fold to about 10 fold, from about2 fold to about 8 fold, from about 2 fold to about 6 fold, from about 2fold to about 4 fold, or about 2 fold.

The nanoparticles of the present invention show unexpected in vitro andin vivo physicochemical and biological parameters in view of the priorart. For example, the blood residence half-time estimated for theligand-attached nanoparticles (e.g., about 5.5 hrs for cRGD-attachednanoparticles) is substantially longer than that of the correspondingligand (e.g., about 13 minutes for cRGD). Montet et. al. Multivalenteffects of RGD peptides obtained by nanoparticle display. J Med Chem.49, 6087-6093 (2006). Extended blood residence half-times may enhanceprobe bioavailability, facilitate tumor targeting, and yield highertumor uptake over longer time periods. In one embodiment, the tumorresidence half-time for the targeted nanoparticles (i.e.,ligand-attached nanoparticles) is about 13 times greater than bloodresidence half-time, whereas the tumor residence half-time for thenon-targeted nanoparticles (i.e., corresponding nanoparticles notattached with ligands) is only about 5 times greater than bloodresidence half-time. This difference suggests substantially greatertumor tissue accumulation of the targeted nanoparticles compared withthe non-targeted nanoparticles. In certain embodiments, given the numberof ligands attached to the nanoparticle, the present nanoparticles showunexpected high-affinity binding (e.g., K_(d) 0.51 nM and IC₅₀ 1.2 nMfor cRGD-attached nanoparticle), multivalency enhancement (e.g., morethan 2 fold enhancement for cRGD-attached nanoparticles compared to cRGDpeptide alone), significant differential tumor uptake (e.g.,cRGD-attached PEG-nanoparticles show about 3 to 4 fold increase indifferential tumor uptake relative to the PEG-coated nanoparticles over72 hrs post-administration), and significant tumor contrast relative tonormal muscle (e.g., about 3 to 5 fold over 72 hrs post-administration)based on tumor-to-muscle uptake ratios.

In one embodiment, three-fold activity-concentration increases werefound for ligand-attached nanoparticles in integrin-expressing tumorsover controls (e.g., ligand-attached nanoparticles in non-integrinexpressing tumors, or corresponding nanoparticles not attached withligands in integrin-expressing tumors) at the time of maximum tumoruptake (about 4 hrs post-injection of the nanoparticles). In addition,tumor-to-muscle uptake ratios for targeted nanoparticles (i.e.,ligand-attached nanoparticles) reveal enhanced tumor tissue contrastrelative to normal muscle, compared with decreased tumor tissue contrastrelative to normal muscle for non-targeted nanoparticles (i.e.,corresponding nanoparticles not attached with ligands), suggesting thatthe targeted nanoparticles are tumor-selective.

In another embodiment, the targeted and non-targeted nanoparticles bothshow efficient renal excretion over the same time period. Nearly half ofthe injected dose is excreted over the first 24 hrs post-injection andabout 72% by 96 hrs, suggesting that the bulk of excretion occurred inthe first day post-injection. By contrast, fecal excretion profiles ofthe targeted nanoparticles indicate that, on average, 7% and 15% of theinjected dose is eliminated over 24 and 96 hrs, respectively.

The physicochemical and biological parameters of the non-toxicnanoparticles, along with its multimodal imaging capabilities (e.g., PETand optical imaging), expand the range of their potential biomedicalapplications. The applications include (a) long-term monitoring: theextended blood circulation time and corresponding bioavailability of thenanoparticles highlight their versatility for both early and long-termmonitoring of various stages of disease management (such as diagnosticscreening, pre-treatment evaluation, therapeutic intervention, andpost-treatment monitoring) without restrictions imposed by toxicityconsiderations; (b) improved tumor penetration: the clearance propertiesof the targeted nanoparticles (e.g., their renal clearance is slowerthat of the molecular probes in the prior art) will be useful forvarious types of biological applications. For example, the nanoparticleswould be particularly useful in cases of poorly vascularized andrelatively inaccessible solid tumors in which localization of agents istypically slow after systemic administration; (c) multimodal imagingcapabilities: these modalities can be combined at multiple scales (i.e.,whole body to cellular levels) for acquiring complementary,depth-sensitive biological information. For example, in SLN mapping,deep nodes can be mapped by PET in terms of their distribution andnumber, while more precise and detailed localization of superficialnodes can be obtained by fluorescence imaging; and (d) targetedtherapeutics: longer clearance of the targeted nanoparticles from tumorcompared to that from blood may be exploited for combineddiagnostic/therapeutic applications, in which the nanoparticles canserve as a radiotherapeutic or drug delivery vehicle.

The present invention further provides a pharmaceutical compositioncomprising the present nanoparticle. The pharmaceutical compositions ofthe invention may be administered orally in the form of a suitablepharmaceutical unit dosage form. The pharmaceutical compositions of theinvention may be prepared in many forms that include tablets, hard orsoft gelatin capsules, aqueous solutions, suspensions, and liposomes andother slow-release formulations, such as shaped polymeric gels.

Suitable modes of administration for the present nanoparticle orcomposition include, but are not limited to, oral, intravenous, rectal,sublingual, mucosal, nasal, ophthalmic, subcutaneous, intramuscular,transdermal, spinal, intrathecal, intra-articular, intra-arterial,sub-arachnoid, bronchial, and lymphatic administration, and other dosageforms for systemic delivery of active ingredients. The presentpharmaceutical composition may be administered by any method known inthe art, including, without limitation, transdermal (passive via patch,gel, cream, ointment or iontophoretic); intravenous (bolus, infusion);subcutaneous (infusion, depot); transmucosal (buccal and sublingual,e.g., orodispersible tablets, wafers, film, and effervescentformulations; conjunctival (eyedrops); rectal (suppository, enema)); orintradermal (bolus, infusion, depot). The composition may be deliveredtopically.

Oral liquid pharmaceutical compositions may be in the form of, forexample, aqueous or oily suspensions, solutions, emulsions, syrups orelixirs, or may be presented as a dry product for constitution withwater or other suitable vehicle before use. Such liquid pharmaceuticalcompositions may contain conventional additives such as suspendingagents, emulsifying agents, non-aqueous vehicles (which may includeedible oils), or preservatives.

The nanoparticle pharmaceutical compositions of the invention may alsobe formulated for parenteral administration (e.g., by injection, forexample, bolus injection or continuous infusion) and may be presented inunit dosage form in ampules, pre-filled syringes, small volume infusioncontainers or multi-dose containers with an added preservative. Thepharmaceutical compositions may take such forms as suspensions,solutions, or emulsions in oily or aqueous vehicles, and may containformulating agents such as suspending, stabilizing and/or dispersingagents. Alternatively, the pharmaceutical compositions of the inventionmay be in powder form, obtained by aseptic isolation of sterile solid orby lyophilization from solution, for constitution with a suitablevehicle, e.g., sterile, pyrogen-free water, before use.

For topical administration to the epidermis, the pharmaceuticalcompositions may be formulated as ointments, creams or lotions, or asthe active ingredient of a transdermal patch. Suitable transdermaldelivery systems are disclosed, for example, in A. Fisher et al. (U.S.Pat. No. 4,788,603), or R. Bawa et al. (U.S. Pat. Nos. 4,931,279;4,668,506; and 4,713,224). Ointments and creams may, for example, beformulated with an aqueous or oily base with the addition of suitablethickening and/or gelling agents. Lotions may be formulated with anaqueous or oily base and will in general also contain one or moreemulsifying agents, stabilizing agents, dispersing agents, suspendingagents, thickening agents, or coloring agents. The pharmaceuticalcompositions can also be delivered via ionophoresis, e.g., as disclosedin U.S. Pat. Nos. 4,140,122; 4,383,529; or 4,051,842.

Pharmaceutical compositions suitable for topical administration in themouth include unit dosage forms such as lozenges comprising apharmaceutical composition of the invention in a flavored base, usuallysucrose and acadia or tragacanth; pastilles comprising thepharmaceutical composition in an inert base such as gelatin and glycerinor sucrose and acacia; mucoadherent gels, and mouthwashes comprising thepharmaceutical composition in a suitable liquid carrier.

For topical administration to the eye, the pharmaceutical compositionscan be administered as drops, gels (S. Chrai et al., U.S. Pat. No.4,255,415), gums (S. L. Lin et al., U.S. Pat. No. 4,136,177) or via aprolonged-release ocular insert (A. S. Michaels, U.S. Pat. No. 3,867,519and H. M. Haddad et al., U.S. Pat. No. 3,870,791).

When desired, the above-described pharmaceutical compositions can beadapted to give sustained release of a therapeutic compound employed,e.g., by combination with certain hydrophilic polymer matrices, e.g.,comprising natural gels, synthetic polymer gels or mixtures thereof.

Pharmaceutical compositions suitable for rectal administration whereinthe carrier is a solid are most preferably presented as unit dosesuppositories. Suitable carriers include cocoa butter and othermaterials commonly used in the art, and the suppositories may beconveniently formed by admixture of the pharmaceutical composition withthe softened or melted carrier(s) followed by chilling and shaping inmolds.

Pharmaceutical compositions suitable for vaginal administration may bepresented as pessaries, tampons, creams, gels, pastes, foams or sprayscontaining, in addition to the nanoparticles and the therapeutic agent,such carriers are well known in the art.

For administration by inhalation, the pharmaceutical compositionsaccording to the invention are conveniently delivered from aninsufflator, nebulizer or a pressurized pack or other convenient meansof delivering an aerosol spray. Pressurized packs may comprise asuitable propellant such as dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol, the dosageunit may be determined by providing a valve to deliver a metered amount.

Alternatively, for administration by inhalation or insufflation, thepharmaceutical compositions of the invention may take the form of a drypowder composition, for example, a powder mix of the pharmaceuticalcomposition and a suitable powder base such as lactose or starch. Thepowder composition may be presented in unit dosage form in, for example,capsules or cartridges or, e.g., gelatin or blister packs from which thepowder may be administered with the aid of an inhalator or insufflator.

For intra-nasal administration, the pharmaceutical compositions of theinvention may be administered via a liquid spray, such as via a plasticbottle atomizer. Typical of these are the Mistometer® (isoproterenolinhaler-Wintrop) and the Medihaler® (isoproterenol inhaler—Riker).

Pharmaceutical compositions of the invention may also contain otheradjuvants such as flavorings, colorings, anti-microbial agents, orpreservatives.

It will be further appreciated that the amount of the pharmaceuticalcompositions required for use in treatment will vary not only with thetherapeutic agent selected but also with the route of administration,the nature of the condition being treated and the age and condition ofthe patient and will be ultimately at the discretion of the attendantphysician or clinician. For evaluations of these factors, see J. F.Brien et al., Europ. J. Clin. Pharmacol., 14, 133 (1978); andPhysicians' Desk Reference, Charles E. Baker, Jr., Pub., MedicalEconomics Co., Oradell, N.J. (41st ed., 1987). Generally, the dosages ofthe therapeutic agent when used in combination with the fluorescentnanoparticles of the present invention can be lower than when thetherapeutic agent is administered alone or in conventionalpharmaceutical dosage forms. The high specificity of the fluorescentnanoparticle for a target site, such as a receptor situated on a cell'ssurface, can provide a relatively highly localized concentration of atherapeutic agent, or alternatively, a sustained release of atherapeutic agent over an extended time period.

The present nanoparticles or compositions can be administered to asubject. The subject can be a mammal, preferably a human. Mammalsinclude, but are not limited to, murines, rats, rabbits, simians,bovines, ovine, swine, canines, feline, farm animals, sport animals,pets, equine, and primates.

The present invention further provides a method for detecting acomponent of a cell comprising the steps of: (a) contacting the cellwith a fluorescent silica-based nanoparticle comprising a silica-basedcore comprising a fluorescent compound positioned within thesilica-based core; a silica shell surrounding at least a portion of thecore; an organic polymer attached to the nanoparticle; from about 1 toabout 20 ligands attached to the nanoparticle; and a contrast agent or achelate attached to the nanoparticle; and (b) monitoring the binding ofthe nanoparticle to the cell or a cellular component (and/or itspotential intracellular uptake) by at least one imaging technique. Theimaging technique may be PET, SPECT, CT, MRI, optical bioluminescence orfluorescence imaging, and combinations thereof.

The location of the cellular component can be detected and determinedinside a metabolically active whole cell, in a whole cell lysate, in apermeabilized cell, in a fixed cell, or with a partially purified cellcomponent in a cell-free environment. The amount and the duration of thecontacting can depend, for example, on the diagnostic or therapeuticobjectives of the treatment method, such as fluorescent detection ofupregulated signaling pathway intermediates (i.e., Akt, NF-κB), diseasestates or conditions, the delivery of a therapeutic agent, or both. Theamount and the duration of the contacting can also depend on therelative concentration of the fluorescent nanoparticle to the targetanalyte, and the state of the cell for treatment.

The present invention further provides a method for targeting a tumorcell comprising administering to a cancer patient an effective amount ofa fluorescent silica-based nanoparticle comprising a silica-based corecomprising a fluorescent compound positioned within the silica-basedcore; a silica shell surrounding at least a portion of the core; anorganic polymer attached to the nanoparticle; a ligand attached to thenanoparticle and capable of binding a tumor marker; and at least onetherapeutic agent. The nanoparticle may be radiolabeled. Thenanoparticle may be administered to the patient by, but not restrictedto, the following routes: oral, intravenous, nasal, subcutaneous, local,intramuscular or transdermal.

In certain embodiments it may be desirable to use a mixture of two ormore types of fluorescent nanoparticles having different properties,such as different fluorescent compound, ligands, organic polymercoatings, contrast agents, or therapeutic agents in order to exploit thebenefits of targeting different components of a tumor cell or differentpopulations of the tumor cells, for example, simultaneously orsequentially.

The methods and compositions of the invention can be used to help aphysician or surgeon to identify and characterize areas of disease, suchas cancers and inflammatory/infectious processes, including, but notrestricted to, cancers of the skin (melanoma), head & neck, prostate,brain, and bowels, to distinguish diseased and normal tissue, such asdetecting tumor margins that are difficult to detect using an ordinaryoperating microscope, e.g., in brain surgery, to help dictate atherapeutic or surgical intervention, e.g., by determining whether alesion is cancerous and should be removed or non-cancerous and leftalone, or in surgically staging a disease, e.g., intraoperative lymphnode staging, sentinel lymph node (SLN) mapping, e.g., nerve-sparingprocedures for preserving vital neural structures (intraparotid nerves).

The methods and compositions of the invention may be used in metastaticdisease detection, treatment response monitoring, SLN mapping/targeting,nerve sparing procedures, residual disease detection, targeted deliveryof therapeutics (combined diagnostic/therapeutic platform), localdelivery of non-targeted, drug-bearing nanoparticles (catheterdelivery), blood-brain barrier therapeutics, treatment ofinflammatory/ischemic diseases (i.e., brain, heart, urinary tract,bladder), combined treatment and sensing of disease (e.g., RatiometricpH sensing, oxygen sensing), etc.

The methods and compositions of the invention can also be used in thedetection, characterization and/or determination of the localization ofa disease, especially early disease, the severity of a disease or adisease-associated condition, the staging of a disease, and/ormonitoring a disease. The presence, absence, or level of an emittedsignal can be indicative of a disease state. The methods andcompositions of the invention can also be used to monitor and/or guidevarious therapeutic interventions, such as surgical and catheter-basedprocedures, and monitoring drug therapy, including cell based therapies.The methods of the invention can also be used in prognosis of a diseaseor disease condition. Cellular subpopulations residing within ormarginating the disease site, such as stem-like cells (“cancer stemcells”) and/or inflammatory/phagocytic cells may be identified andcharacterized using the methods and compositions of the invention. Withrespect to each of the foregoing, examples of such disease or diseaseconditions that can be detected or monitored (before, during or aftertherapy) include cancer (for example, melanoma, thyroid, colorectal,ovarian, lung, breast, prostate, cervical, skin, brain,gastrointestinal, mouth, kidney, esophageal, bone cancer), that can beused to identify subjects that have an increased susceptibility fordeveloping cancer and/or malignancies, i.e., they are predisposed todevelop cancer and/or malignancies, inflammation (for example,inflammatory conditions induced by the presence of cancerous lesions),cardiovascular disease (for example, atherosclerosis and inflammatoryconditions of blood vessels, ischemia, stroke, thrombosis), dermatologicdisease (for example, Kaposi's Sarcoma, psoriasis), ophthalmic disease(for example, macular degeneration, diabetic retinopathy), infectiousdisease (for example, bacterial, viral, fungal and parasitic infections,including Acquired Immunodeficiency Syndrome), immunologic disease (forexample, an autoimmune disorder, lymphoma, multiple sclerosis,rheumatoid arthritis, diabetes mellitus), central nervous system disease(for example, a neurodegenerative disease, such as Parkinson's diseaseor Alzheimer's disease), inherited diseases, metabolic diseases,environmental diseases (for example, lead, mercury and radioactivepoisoning, skin cancer), bone-related disease (for example,osteoporosis, primary and metastatic bone tumors, osteoarthritis) and aneurodegenerative disease.

The methods and compositions of the invention, therefore, can be used,for example, to determine the presence and/or localization of tumorand/or co-resident stem-like cells (“cancer stem cells”), the presenceand/or localization of inflammatory cells, including the presence ofactivated macrophages, for instance in peritumoral regions, the presenceand in localization of vascular disease including areas at risk foracute occlusion (i.e., vulnerable plaques) in coronary and peripheralarteries, regions of expanding aneurysms, unstable plaque in carotidarteries, and ischemic areas. The methods and compositions of theinvention can also be used in identification and evaluation of celldeath, injury, apoptosis, necrosis, hypoxia and angiogenesis.PCT/US2006/049222.

The following examples are presented for the purposes of illustrationonly and are not limiting the invention.

Example 1 Preparation and Characterization of PEG-Coated Nanoparticles

Nanoparticles containing an NIR-emitting dye (Cy-5) were synthesized andfunctionalized by PEGylation according to well-established protocols asdisclosed in PCT/US2008/074894 and Stober et al. Controlled growth ofmonodispersed silica spheres in the micron size range. Colloid InterfaceSci. 1968; 26:62-69. Ohnishi et al. J. Mol. Imaging 2005, 4:172-181. Cy5malemide was reacted with a co-reactive organo silane compound,(3-Mercaptopropyl)tromethoxysilane to form a fluorescent silicaprecursor. This fluorescent silica precursor was co-condensed withtetraethylorthosilicate to form a fluorescent silica based core. APEG-silane compound, with methoxy-terminated poly(ethylene glycol)chains (PEG, ˜0.5 kDa) Methoxy(Polyethyleneoxy)Propyl]-Trimethoxysilane, was added to the fluorescent silica based coreto form a PEG coating on the core. PEG-coated nanoparticles weredialyzed to physiological saline (0.15M NaCl in H2O) through 3500 MWCOSnakeskin Dialysis Membranes and sterile-filtered. All samples wereoptical density-matched at their peak absorption wavelength (640 nm)prior to injection. Hydrodynamic size measurements were achieved byDynamic Light Scattering (DLS) and Fluorescence Correlation Spectroscopy(FCS). Briefly, particles dialyzed to water were measured on aBrookhaven Instruments Company 200SM static/DLS system using a HeNelaser (λ=632.8 nm). Due to overlap of the dye absorption with theexcitation source, 15-min integration times were used to achieveacceptable signal-to-noise ratios. For FCS, particles were dialyzed towater, diluted into 0.15M NaCl, and measured on a Zeiss LSM 510 Confocor2 FCS (HeNe 633 nm excitation). The instrument was calibrated for sizeprior to all measurements. Comparison of the relative brightness ofPEGylated nanoparticles with free dye was determined from FCS curves,measured as count rate per molecule/particle.

Example 2 Renal Clearance of PEG Coated Nanoparticles

Fluorescent core-shell silica nanoparticles, having a hydrodynamicradius of about 3 nm, were synthesized. These nanoparticles were foundto be in the 6-10 nm diameter range, as shown by dynamic lightscattering (DLS) results (FIG. 1A). In vivo whole-body NIR fluorescenceimaging of bare (no PEG coat) silica nanoparticles, on the order of 6-nmand 3.3-nm, in nude mice showed considerable renal clearance 45 minpost-injection with a significant accumulation remaining in the liver(FIG. 1B). Eventual excretion into the enterohepatic circulationoccurred during the ensuing 24 h. On the basis of these results,particles were covalently coated with methoxy-terminated poly(ethyleneglycol) chains (PEG, ˜0.5 kDa), per protocols in PCT/US2008/074894, toprevent opsonization and further enhance particle clearance whilemaintaining a small hydrodynamic size. This treatment decreased liverretention and resulted in increased renal filtration into the bladder at45 min post-injection by NIR fluorescence imaging (FIG. 1C), withbladder fluorescence visible out to 24 h. The probes were welltolerated, with no adverse effects or animal deaths observed over thecourse of the study. Serial co-registered PET-CT imaging 24-hr afterinjection of ¹²⁴I-labeled PEG coated nanoparticles (FIG. 1D, upper row)demonstrated a small amount of residual bladder activity, as well asactivity overlying the liver/gastrointestinal tract (center), flanked byindependently acquired microCT and microPET scans. Serial microPETimages confirmed findings on NIR fluorescence imaging. The half-time ofblood residence of the ¹²⁴I-labeled PEGylated nanoparticles based ontime-dependent changes in blood activity over a 96-hour period was foundto be 7.3 hours. For the ¹²⁴I-labeled, RGD-bound nanoparticles, thehalf-time of blood residence was found to be 5.6 hours.

Based on these in vivo data, a more detailed biodistribution andclearance study of coated nanoparticles was undertaken on two sets ofPEGylated Cy5-containing particles to assess the effects of probe sizeon biodistribution. Nanoparticles with hydrodynamic diameters of3.3±0.06 and 6.0±0.1 nm, as measured by fluorescence correlationspectroscopy (FCS), were generated (FIG. 2A). Prior to in vivo studies,particle photophysical properties were investigated to establish theirperformance levels versus free dye. Despite the extremely small particlesize, silica-encapsulated dye molecules exhibited photophysicalenhancements over free dye that scaled with particle size, includingsignificant increases in brightness, as determined by absorption andemission spectroscopy (FIG. 2B) and FCS (FIG. 2C). Compared to the freedye, the 3.3 and 6.0 nm diameter nanoparticles exhibited 2- and 3-foldincreases in photobleaching half-life, respectively, when irradiatedwith a high power 635 nm laser (FIG. 2D). Thus, these nanoparticleprobes were found to be both brighter and more photostable than theirfree dye counterparts.

In addition to semiquantitative evaluation of in vivo nanoparticlebehavior from whole-body imaging, ex-vivo analysis of tissue homogenatesand fluids was performed using a fluorescence plate reader, whichallowed calibrated quantitation of variations observed in NIRfluorescence imaging. Samples were grouped as “retained” (liver, kidney,lung, spleen homogenates, and blood) and “excreted” (urine) sources ofparticle fluorescence, were background-corrected and were converted topercent of the initial dose (% ID) per animal based on calibrationcurves. Tissue analysis showed minimal particle retention in majororgans, with most of the fluorescence attributed to circulating blood(FIG. 3A). Net particle retention, calculated as the sum of the“retained” components, was fit with an exponential decay curve todetermine the kinetics of excretion (FIG. 3B). Larger particlesexhibited a longer tissue half-life (t_(1/2) (3.3 nm)=190 min, t_(1/2)(6.0 nm)=350 min) and greater initial organ retention. After 48 h, the6-nm particle exhibited minimal retention in the body (R_(total) (6.0nm)=2.4±0.6% ID). Urine samples collected at the time of sacrifice, inconjunction with serial dilution calibration data, was used to estimatethe total renal clearance based on a conservative estimate of theaverage urine volume excreted per unit time. By this method, the % IDexcreted over time for both particle sizes (FIG. 3C) was estimated.

Example 3 Fluorescent Silica Nanoparticles Conjugated with α_(v)β₃Integrin-Targeting Peptide (Melanoma Model)

To synthesize a multimodal nanoparticle with high affinity for tumormarker α_(v)β₃ integrin, linear RGD hexapeptide (CGGRGD) was conjugatedto the nanoparticle via a Cys-maleimide linkage. Male athymic nude micewere injected subcutaneously into their flanks with C6 rat glioma cells.At ˜0.5 cm in diameter, mice were IV-injected with either bare silicananoparticles or PEG-ylated RGD nanoparticles (˜500 nm/kg). FIGS. 4A-4Cshow the in vivo biodistribution in non-tumor-bearing and tumor-bearingmice.

In vitro binding characteristics of targeted (RGD-bound) andnon-targeted (PEG-coated) nanoparticles to α_(v)β₃-integrin-positive(M21 cells) and integrin-negative (M21L cells) human melanoma cell lineswere investigated using flow cytometry (FIGS. 5A, 5B).

Example 4 Fluorescent Silica Nanoparticles Conjugated with α_(v)β₃Integrin-Targeting Peptide and Nodal Mapping (Melanoma Model)

We utilized a biocompatible material, silica, which has an architecturethat could be precisely tuned to particle sizes optimized for renalclearance. We attached small targeting peptides and a radioactive labelto the particle surface for serial PET imaging measurements in awell-characterized in vivo human melanoma model, and mapped draininglymph nodes and lymphatic channels using an encapsulated near infrared(NIR) dye and multi-scale optical fluorescence imaging methods. Ballouet al., Sentinel lymph node imaging using quantum dots in mouse tumormodels. Bioconjugate Chem. 18, 389-396 (2007). Kim et al., Near-infraredfluorescent type II quantum dots for sentinel lymph node mapping. Nat.Biotechnol. 22, 93-97 (2003). Tanaka et al, Image-guided oncologicsurgery using invisible light: completed pre-clinical development forsentinel lymph node mapping. J Surg Oncol. 13, 1671-1681 (2006).Toxicity testing was also performed and human normal-organ radiationdoses derived. Specifically, we synthesized ˜7 nm diameter,near-infrared (NIR) dye-encapsulating core-shell silica nanoparticles,coated with PEG chains and surface functionalized with a small number(˜6-7) of targeting peptides and radiolabels.

We demonstrate that these probes simultaneously are non-toxic, exhibithigh-affinity/avidity binding, efficient excretion, and significantdifferential uptake and contrast between tumor and normal tissues usingmultimodal molecular imaging approaches. The sensitive detection,localization, and interrogation of lymph nodes and lymphatic channels,enabled by the NIR dye fluorescence, highlights the distinct potentialadvantage of this multimodal platform for detecting and stagingmetastatic disease in the clinical setting, while extending the lowerrange of nodal sizes that can be detected.

Materials and Methods

Synthesis of cRGDY-PEG-Nanoparticles and PEG-Nanoparticles

Particles were prepared by a modified Stober-type silica condensation asdescribed previously. Wiesner et al., Peg-coated Core-shell SilicaNanoparticles and Mathods of Manufactire and Use, PCT/US2008/74894.Larson, et al., Silica nanoparticle architecture determines radiativeproperties of encapsulated chromophores. Chem. Mater. 20, 2677-2684(2008). Bogush, et al., Preparation of Monodisperse Silica Particles:Control of Size and Mass Fraction. J. Non-Cryst. Solids, 104, 95-106(1988). Sadasivan, et al., Alcoholic Solvent Effect on SilicaSynthesis—NMR and DLS Investigation. J. Sol-Gel Sci. Technol. 12, 5-14(1998). Herz, et al., Large Stokes-Shift Fluorescent SilicaNanoparticles with Enhanced Emission over Free Dye for Single ExcitationMultiplexing. Macromol Rapid Commun. 30, 1907-1910 (2009). Tyrosineresidues were conjugated to PEG chains for attachment of radioiodine orstable iodine moieties. Hermanson, Bioconjugate Techniques, (AcademicPress, London, ed. 2, 2008). All samples were optical density-matched attheir peak absorption wavelength (640 nm) prior to radiolabeling. cRGDpeptides were attached to functionalized PEG chains via acysteine-maleimide linkage, and the number of cRGD peptides bound to theparticle was estimated using FCS-based measurements of absolute particleconcentrations and the starting concentration of the reagents for cRGDpeptide.

Hydrodynamic Size and Relative Brightness Comparison Measurements byFluorescence Correlation Spectroscopy (FCS) Particles dialyzed to waterwere diluted into physiological saline (0.15 M NaCl in H₂O) and measuredon a Zeiss LSM 510 Confocor 2 FCS using HeNe 633-nm excitation. Theinstrument was calibrated for size prior to all measurements. Diffusiontime differences were used to evaluate variations in the hydrodynamicsizes of the dye and particle species. Relative brightness comparisonsof the free dye and the PEG- and the RGDY-PEG nanoparticles wereperformed using count rates per molecule/particle.

Radiolabeling of C Dot Conjugates

Radiolabeling of the cRGDY-PEG and PEG-nanoparticles was performed usingthe IODOGEN method (Pierce, Rockford, Ill.). Piatyszek, et al., Iodo-genmediated radioiodination of nucleic acids. J. Anal. Biochem. 172,356-359 (1988). Activities were measured by gamma (γ)-counting andfluorescence measured using a Varian fluorometer (excitation 650nm/emission 680).

Cells and Cell Culture

Human melanoma M21 and M21 variant (M21-L, α_(v) negative) cell lineswere maintained in RPMI 1640 media/10% fetal BSA, 2 mM L-glutaminepenicillin, and streptomycin (Core Media Preparation Facility, MemorialSloan Kettering Cancer Center, New York). Human umbilical venous cordendothelial cells (HUVECs) were cultured in M199 media/10% fetal bovineserum, 20 μg/ml endothelial cell growth factor, 50 μg/ml heparin,penicillin and streptomycin.

In Vitro Cell-Binding and Molecular Specificity of¹²⁴I-cRGD-PEG-Nanoparticles

To assay particle binding and specificity for M21 cells, 24-well plateswere coated with 10 μg/ml collagen type I (BD Biosciences, Bedford,Mass.) in phosphate buffered saline (PBS) and incubated (37° C., 30min). M21 cells (3.0-4.0×105 cells/well) were grown to confluency andwashed with RPMI 1640 media/0.5% bovine serum albumin (BSA). ¹²⁴I-cRGD-PEG-nanoparticles (0-4.0 ng/ml) were added to wells and cellsincubated (25° C., 4 hours), washed with RPMI 1640 media/0.5% BSA, anddissolved in 0.2 M NaOH. Radioactivity was assayed using a 1480Automatic Gamma Counter (Perkin Elmer) calibrated for iodine-124.Nonspecific binding was determined in the presence of a 1000-fold excessof cRGD (Peptides International, Louisville, Ky.). Scatchard plots ofthe binding data were generated and analyzed using linear regressionanalyses (Microsoft Excel 2007) to derive receptor-binding parameters(Kd, Bmax, IC50).

In Vitro Cell-Binding Studies Using Optical Detection Methods

Maximum differential binding of cRGDY-PEG-nanoparticles andPEG-nanoparticles to M21 cells was determined for a range of incubationtimes and particle concentrations using flow cytometry, with optimumvalues used in competitive binding and specificity studies. Cells(3.0×10⁵ cells/well) were washed with RPMI 1640 media/0.5% BSA, detachedusing 0.25% trypsin/EDTA, and pelleted in a microcentrifuge tube (5 minat 1400 rpm, 25° C.). Pellets were resuspended in BD FACSFlow solution(BD Biosciences, San Jose, Calif.) and analyzed in the Cy5 channel todetermine the percentage of particle-bound probe (FACSCalibur, BectonDickinson, Mountain View, Calif.). Competitive binding studies wereadditionally performed following incubation of cRGDY-PEG-nanoparticles(2.5 ng/ml) with M21, M21L, and HUVEC cells in the presence of excesscRGD and/or mouse monoclonal anti-human integrin α_(v)β₃fluorescein-conjugated antibody (Millipore, Temecula, Calif.) andanalyzed by flow cytometry. To assess potency of the RGDY-PEGnanoparticles relative to the cRGD peptide, anti-adhesion assays wereperformed. Ninety-six-well microtiter plates were coated withvitronectin in PBS (5 μg/ml), followed by 200 μl of RPMI/0.5% BSA (1 h,37° C.). Cells (3×10⁴/100 μl/well) were incubated in quadruplicate (30min, 25° C.) with various concentrations of cRGDY-PEG-nanoparticles orcRGD peptide in RPMI/0.1% BSA, and added to vitronectin-coated plates(30 min, 37° C.). Wells were gently rinsed with RPMI/0.1% BSA to removenon-adherent cells; adherent cells were fixed with 4% PFA (20 min, 25°C.) and stained with methylene blue (1 h, 37° C.) for determination ofoptical densities, measured using a Tecan Safire plate reader (λex=650nm, λem=680 nm, 12 nm bandwidth). The multivalent enhancement factor wascomputed as the ratio of the cRGD peptide to cRGDY-PEG-dot IC50 values.Montet, et al., Multivalent effects of RGD peptides obtained bynanoparticle display. J Med Chem. 49, 6087-6093 (2006).

Animal Models and Tumor Innoculation

All animal experiments were done in accordance with protocols approvedby the Institutional Animal Care and Use Committee of MemorialSloan-Kettering Cancer Center and followed National Institutes of Healthguidelines for animal welfare. Male athymic nu/nu mice (6-8 weeks old,Taconic Farms Inc, Hudson, N.Y.) were provided with water containingpotassium iodide solution to block uptake by the thyroid gland of anyfree radioiodine in vivo, and maintained on a Harlan Teklad Global Diet2016, ad libitum, as detailed elsewhere 10. To generate M21 or M21Lxenografts, equal volumes of cells (˜5×10⁶/100 μl) and matrigel wereco-injected subcutaneously into the hindleg in different mice. Tumorsizes were regularly measured with calipers, yielding average tumorvolumes of 200 mm³.

In Vivo Pharmacokinetic and Residence Half-Time (T_(1/2)) Measurements

Time-dependent activity concentrations (% ID/g), corrected forradioactive decay to the time of injection, were measured by sacrificinggroups of mice at specified times following i.v. injection of¹²⁴I-cRGDY-PEG-nanoparticles or ¹²⁴I-PEG-nanoparticles (˜20 μCi/mouse)and harvesting, weighing, and counting blood, tumor, and organs in ascintillation γ-counter calibrated for ¹²⁴I. The resulting time-activityconcentration data for each tissue were fit to a decreasingmonoexponential function to determine the values of T_(1/2) and A, thetissue/organ residence half time and zero-time intercept, respectively,of the function.

The fraction of particles excreted in the urine over time was estimatedusing previously described methods. Burns, et al., Fluorescent SilicaNanoparticles with Efficient Urinary Excretion for Nanomedicine, NanoLetters 9, 442-8 (2009). Briefly, mice were injected i.v. with either200 μl unlabeled cRGDY-PEG-nanoparticles or PEG-nanoparticles, and urinesamples collected over a 168-hr period (n=3 mice per time point).Particle concentrations at each time point were determined usingfluorometric analyses and a serial dilution calibration curve generatedfrom background-corrected fluorescence signal measurements of urinesamples mixed with known particle concentrations (% ID). Concentrationvalues, along with estimates of average daily mouse urine volumes, werethen used to compute the cumulative % ID/g urine excreted over time. Toassess cumulative fecal excretion, metabolic cages were used to collectfeces over a similar time interval after i.v. injection of 200 μl¹²⁴I-cRGDY-PEG-nanoparticles (n=4 mice per time point). Specimenactivities were measured using a γ-counter calibrated for ¹²⁴I.

Dosimetry

Time-activity functions derived for each tissue were analyticallyintegrated (with inclusion of the effect of radioactive decay) to yieldthe corresponding cumulative activity (i.e. the total number ofradioactive decays). ¹²⁴I mouse organ absorbed doses were thencalculated by multiplying the cumulative activity by the ¹²⁴Iequilibrium dose constant for non-penetrating radiations (positrons),assuming complete local absorption of such radiations and ignoring thecontribution of penetrating radiations (i.e., γ-rays). Eckerman, et al.,Radionuclide Data and Decay Schemes, 2nd ed. Reston, Va.: Society ofNuclear Medicine; 1989. The mouse normal-organ cumulated activities wereconverted to human normal-organ cumulated activities by adjustment forthe differences in total-body and organ masses between mice and humans(70-kg Standard Man). Cristy, et al., Specific absorbed fractions ofenergy at various ages from internal photon sources (I-VII). Oak RidgeNational Laboratory Report ORNL/TM-8381/V1-7. Springfield, Va.: NationalTechnical Information Service, Dept of Commerce; 1987. The humannormal-organ cumulated activities calculated were entered into theOLINDA dosimetry computer program to calculate, using the formalism ofthe Medical Internal Dosimetry (MIRD) Committee of the Society ofNuclear Medicine, the Standard-Man organ absorbed doses. Loevinger, etal., MIRD Primer for Absorbed Dose Calculations (Society of NuclearMedicine, New York, 1991). Stabin, et al., OLINDA/EXM: thesecond-generation personal computer software for internal doseassessment in nuclear medicine. J Nucl Med. 46, 1023-1027 (2005).

Acute Toxicity Studies and Histopathology

Acute toxicity testing was performed in six groups of male and femaleB6D2F1 mice (7 wks old, Jackson Laboratories, Bar Harbor, Me.). Thetreatment group (n=6 males, n=6 females) received unlabeled targetedprobe (¹²⁷I-RGDY-PEG-nanoparticles) and the control group (n=6 males,n=6 females) unlabeled iodinated PEG-nanoparticles (vehicle, ¹²⁷I-RGDY-PEG-nanoparticles) in a single i.v. injection (200 μl). Untreatedcontrols (n=2 males, n=2 females) were additionally tested. Mice wereobserved daily over 14 days p.i. for signs of morbidity/mortality andweight changes, and gross necropsy, histopathology, and blood samplingfor hematology and serum chemistry evaluation was performed at 7- and14-days p.i (FIGS. 10A-10B and Table 3).

Serial PET Imaging of Tumor-Specific Targeting

Imaging was performed using a dedicated small-animal PET scanner (Focus120 microPET; Concorde Microsystems, Nashville, Tenn.). Mice bearing M21or M21L hindleg tumors were maintained under 2% isoflurane anesthesia inoxygen at 2 L/min during the entire scanning period. One-hour list-modeacquisitions were initiated at the time of i.v. injection of 200 μCi of¹²⁴I-cRGDY-PEG-nanoparticles or ¹²⁴I-PEG-nanoparticles in all mice,followed by serial 30 min static images over a 96-hour interval. Imagedata were corrected for non-uniformity of the scanner response, deadtime count losses, random counts, and physical decay to the time ofinjection. Voxel count rates in the reconstructed images were convertedto activity concentrations (% ID/g) by use of a measured systemcalibration factor. Three-dimensional region-of-interest (ROI) analysisof the reconstructed images was performed by use of ASIPro software(Concorde Microsystems, Nashville, Tenn.) to determine the mean,maximum, and SD of probe uptake in the tumors. Tumor-to-muscle activityconcentration ratios were derived by dividing the image-derived tumor %ID/g values by the γ-counter muscle % ID/g values.

Nodal Mapping Using Combined NIR Fluorescence Imaging and Microscopy

Nude mice bearing hindleg tumors were injected by 4-quadrant,peritumoral administration using equal volumes of a 50-μ1 cRGDY-PEG-dotsample and allowed to perambulate freely. Following a 30 min to 1-hrinterval, mice were anesthetized with a 2% isofluorine/98% oxygenmixture, and a superficial paramidline incision was made verticallyalong the ventral aspect of the mouse to surgically expose the regionfrom the hindlimb to the axilla ipsilateral to the tumor. In situoptical imaging of locoregional nodes (i.e., inguinal, axillary) anddraining lymphatics (including axillary region) was performed using amacroscopic fluorescence microscope fitted with 650±20 nm NIR excitationand 710-nm long-pass emission filters. Whole-body optical images(Cambridge Research Instruments Maestro imager) were additionallyacquired and spectrally deconvolved as reported previously. Burns, etal., Fluorescent Silica Nanoparticles with Efficient Urinary Excretionfor Nanomedicine, Nano Letters 9, 442-8 (2009).

Statistical Analysis

Statistical analyses comparing groups of tumor mice receivingtargeted/non-targeted probes or bearing M21/M21L tumors, were performedusing a one-tail Mann-Whitney U test, with P<0.05 consideredstatistically significant. For biodistribution studies, thetissue-specific mean % ID/g values of ¹²⁴I-cRGDY-PEG-(n=7 mice) and¹²⁴I-PEG-nanoparticles (control, n=5 mice) were compared at each timepoint, with statistically significant differences in tracer activitiesobserved in blood, tumor, and major organs at 4 and 96 hrs p.i., as wellas at 24 hrs p.i. for tumor and other tissues (Table 1). For tumortargeting studies, differences in mean % ID/g values between M21 (n=7)and M21L tumor mice (n=5), as well as mice receiving control probes(n=5), were found to be maximal at 4 hrs p.i. (p=0.0015 for bothcontrols), remaining significantly elevated at 24 hrs (p=0.0015 andp=0.004, respectively), 48 hrs (p=0.001 and p=0.003, respectively), 72hrs (p=0.015 and 0.005, respectively), and 96 hrs (p=0.005 forM21-M21L). Tumor-to-muscle ratios for ¹²⁴I-cRGDY-PEG-nanoparticles (n=7)versus ¹²⁴I-PEG-nanoparticles (n=5) were found to be statisticallysignificant at 24 hrs p.i. (p=0.001) and 72 hrs p.i. (p=0.006), but notat 4 hrs p.i. (p=0.35). Goodness of fit values (R2), along with theirassociated p values, were determined for the urine calibration curve(R2=0.973, p=0.01), as well as for the urine (R2>0.95, p=0.047) andfecal (R2>0.995, p<0.002) cumulative % ID excretion curves usingnon-linear regression analyses (SigmaPlot, Systat, v. 11.0).

Results Nanoparticle Design and Characterization

Cy5 dye encapsulating core-shell silica nanoparticles (emissionmaxima >650 nm), coated with methoxy-terminated polyethylene glycol(PEG) chains (PEG ˜0.5 kDa), were prepared according to previouslypublished protocols. Burns, et al., Fluorescent Silica Nanoparticleswith Efficient Urinary Excretion for Nanomedicine, Nano Letters, 9,442-8 (2009). Ow, et al., Bright and stable core-shell fluorescentsilica nanoparticles. Nano Lett. 5, 113-117 (2005). The neutral PEGcoating prevented non-specific uptake by the reticuloendothelial system(opsonization). The use of bifunctional PEGs enabled attachment of smallnumbers (˜6-7 per particle) of α_(v)β₃ integrin-targeting cyclicarginine-glycine-aspartic acid (cRGDY) peptide ligands to maintain asmall hydrodynamic size facilitating efficient renal clearance. Peptideligands were additionally labeled with ¹²⁴I through the use of atyrosine linker to provide a signal which can be quantitatively imagedin three dimensions by PET (¹²⁴I-cRGDY-PEG-dots, FIG. 6A); an importantpractical advantage of relatively long-lived ¹²⁴I (physical half-life:4.2 d) is that sufficient signal persists long enough to allowradiodetection up to at least several days postadministration, whenbackground activity has largely cleared and tumor-to-background contrastis maximized. Purity of the radiolabeled targeted nanoparticle was >95%by radio thin layer chromatography. Stability of the non-radiolabeledtargeted nanoparticle is about 1 year by FCS measurements. Particle isexcreted intact in the urine by FCS analyses. As used herein, “dot” and“nanoparticle” are used interchangeably. A PEG-coated particlecontaining a tyrosine residue for ¹²⁴I labeling served as the controlprobe (¹²⁴I-PEG-dots). Purification of the radiolabeled samples by sizeexclusion chromatography (FIG. 7) resulted in radiochemical yieldsof >95%. Hydrodynamic diameters of ˜7 nm i.d. were measured fornon-radioactive cRGDY-PEG-dots and PEG-dots by fluorescence correlationspectroscopy (FCS) (FIGS. 6B and 6C). The relative brightness of thecRGDY-PEG-dots was determined, on average, to be 200% greater than thatof the free dye (FIG. 6C), consistent with earlier results. Burns, etal., Fluorescent Silica Nanoparticles with Efficient Urinary Excretionfor Nanomedicine, Nano Letters, 9, 442-8 (2009). Larson, et al., Silicananoparticle architecture determines radiative properties ofencapsulated chromophores. Chem. Mater. 20, 2677-2684 (2008). Based onthese physicochemical properties, we anticipated achieving a favorablebalance between selective tumor uptake and retention versus renalclearance of the targeted particle, thus maximizing target-tissuelocalization while minimizing normal-tissue toxicity and radiationdoses.

In Vitro Receptor Binding Studies

To examine in vitro binding affinity and specificity of¹²⁴I-cRGDY-PEG-dots and ¹²⁴I-PEGdots to tumor and vascular endothelialsurfaces, α_(v)β₃ integrin-overexpressing (M21) and nonexpressing (M21L)melanoma and human umbilical vein endothelial (HUVECs) cell lines wereused. Highly specific linear and saturable binding of the cRGDY-PEG-dotswas observed over a range of particle concentrations (0 to 8 ng/ml) andincubation times (up to 5-hrs), with maximum differential binding at4-hr and ˜2.0 ng/ml particle concentration (data not shown) using flowcytometry. Receptor-binding specificity of ¹²⁴I-cRGDY-PEG dots wastested using γ-counting methods after initially incubating M21 cellswith excess non-radiolabeled cRGD and then adding various concentrationsof the radiolabeled targeted probe (FIG. 8A). Scatchard analysis of thebinding data yielded a dissociation equilibrium constant, Kd, of 0.51 nM(FIG. 8A, inset) and receptor concentration, Bmax, of 2.5 pM. Based onthe Bmax value, the α_(v)β₃ integrin receptor density was estimated tobe 1.0×10⁴ per M21 cell, in reasonable agreement with the previouslypublished estimate of 5.6×10⁴ for this cell line. Cressman, et al.,Binding and uptake of RGD-containing ligands to cellular α_(v)β₃integrins. Int J Pept Res Ther. 15, 49-59 (2009). Incremental increasesin integrin-specific M21 cellular uptake were also observed over atemperature range of 4 to 37° C., suggesting that receptor-mediatedcellular internalization contributed to overall uptake (data not shown).Additional competitive binding studies using the targeted probe showedcomplete blocking of receptor-mediated binding with anti-α_(v)β₃integrin antibody (FIG. 8B) by flow cytometry. No significant reductionwas seen in the magnitude of receptor binding (˜10% of M21) with M21Lcells (FIG. 8C) using either excess cRGDY or anti-α_(v)β₃ integrinantibody. These results were confirmed by additional γ-counting studies,and a 50% binding inhibition concentration, IC50, of 1.2 nM wasdetermined for the ¹²⁴I-cRGDY-PEG-dot. An associated multivalentenhancement factor of greater than 2.0 was found for the cRGDY-PEG-dotrelative to the monomeric cRGD peptide using an anti-adhesion assay andM21 cells (data not shown). Montet, et al., Multivalent effects of RGDpeptides obtained by nanoparticle display. J Med Chem. 49, 6087-6093(2006). Li, et al., ⁶⁴Cu-labeled tetrameric and octomeric RGD peptidesfor small-animal PET of tumor α_(v)β₃ integrin expression. J. Nucl Med.48, 1162-1171 (2007). Similar to M21 cells, excess antibody effectivelyblocked cRGDY-PEG-dot receptor binding to HUVEC cells by flow cytometry(FIG. 8D).

Biodistribution and Clearance Studies

The time-dependent biodistribution, as well as renal and hepatobiliaryclearance were evaluated by intravenously administering tracer doses(˜0.2 nanomoles) of ¹²⁴I-cRGDY-PEGdots and ¹²⁴I-PEG-dots to M21 tumorxenograft mouse models (FIGS. 9A-9D). Although tissueactivity-concentrations (percent of the injected dose per gram (% ID/g))for the targeted probe were measured over a 196-hr post-injection (p.i.)time interval, comparison of the ¹²⁴I-cRGDY-PEGdot (FIG. 9A) and¹²⁴I-PEG-dot tracers (FIG. 9B) was restricted to a 96-hr window, as datafor the latter was not acquired at 1 week. Statistically significant(p<0.05) differences in tracer activities were observed for blood,tumor, and major organs at 4 and 96 hrs p.i., as well as at 24 hrs p.i.for the tumor and several other tissues (Table 1). The targeted probewas almost entirely eliminated from the carcass at 1 week p.i (˜3% ID).The residence half times (T_(1/2)) for blood, tumor, and major organsfor these tracers are shown in Table 2 (columns 2 and 5). Arepresentative data set (blood residence) is shown in the inset of FIG.9A. A relatively long blood T_(1/2) value of 7.3±1.2 hrs was determinedfor the ¹²⁴I-PEG-dot. Upon attachment of the cRGDY peptide to synthesizethe ¹²⁴I-cRGDY-PEG-dot, the T_(1/2) value decreased slightly to 5.6±0.15hrs, but was accompanied by greater probe bioavailability (Table 2,column 3). The tumor T_(1/2) value for the ¹²⁴I-cRGDY-PEG-dot was foundto be about 13 times greater than that for blood, versus only a 5-folddifference for the ¹²⁴I-PEG-dot (Table 2, columns 2 and 5).

TABLE 1 Biodistribution study p-values comparing ¹²⁴I-cRGDY-PEG- and¹²⁴I-PEG-dots Post-injection times (hours) Tissue 4 24 96 Blood 0.0010.113 0.010 Tumor 0.045 0.012 0.001 Heart 0.019 0.231 0.001 Lungs —0.039 0.006 Liver 0.001 0.033 0.028 Spleen 0.001 0.208 0.001 SmallIntestine 0.001 0.046 0.002 Large Intestine 0.001 0.137 0.003 Kidneys —0.356 0.001 Muscle 0.001 0.007 0.001 Brain 0.001 0.074 0.001

TABLE 2 Mouse Human^(†) ¹²⁴I-RGDY-PEG ¹²⁴I-PEG ¹²⁴I RGDY-PEG ¹²⁴I-PEGT_(1/2) A Absorbed Dose T_(1/2) A Absorbed Dose Absorbed Dose TargetOrgan (h) (% ID/g) (rad/mCi) (h) (% ID/g) (rad/mCi) (rad/mCi) Blood 5.918.8 626 7.3 4.7 189 (see red marrow below) Heart 6.8 7.0 266 34.1 0.8120 0.307 (Wall) 0.087 Lungs 8.5 5.7 267 37.7 3.0 498 0.298 0.263 Liver65.9 3.9 935 57.5 1.4 294 0.486 0.234 Spleen 42.3 45.6 1071 27.4 45.7410 3.20 0.254 195. 286 Small Intestine 30.3 1.8 251 13.2 0.9 61 0.3040.115 Large Intestine 23.9 2.0 228 49.2 0.5 99 0.427 (U) 0.209 0.724 (L)0.416 Kidneys 66.0 3.0 712 33.0 2.0 388 2.50 0.320 Muscle 27.7 0.8 10547.1 0.2 38 0.227 0.060 Brain 13.9 0.4 29 8.5 0.2 8 0.187 0.149^(§)Tumor 73.5 1.5 380 37.0 0.9 146 n/a n/a ^(ζ)Bone (see osteogeniccells) Adrenals 0.400 0.083 Breasts 0.141 0.042 Gallbladder Wall 0.2890.097 Stomach Wall 0.265 0.065 Ovaries 0.303 0.124 Pancreas 0.389 0.081Red Marrow 1.07 0.084 Osteogenic Cells 0.203 0.127 Skin 0.158 0.038Testes 0.186 0.073 Thymus 0.173 0.052 Thyroid 0.188 0.043 UrinaryBladder Wall 2.01 1.65 Uterus 0.333 0.171 Total Body 0.034 0.075Effective Dose Equivalent (rem/mCi) 0.863 0.256 Effective Dose (rem/mCi)0.599 0.232 ^(†)70-kg Standard Man, U (upper), L (lower), ^(§)mousemelanoma model, ^(ζ)bone activity much lower than other tissues (notreported)

By appropriate mass-adjusted translation of the foregoingbiodistribution data to man, human normal-organ radiation doses werederived and found to be comparable to those of other commonly useddiagnostic radiotracers (Table 2, columns 8, 9). Along with the findingthat the targeted probe was non-toxic and resulted in no tissue-specificpathologic effects (i.e., no acute toxicity) (FIGS. 10A-10B and Table3), first-in-man targeted and nontargeted molecular imaging applicationswith these agents are planned.

In another study to confirm that ¹²⁷I-RGD-PEG dots are non-toxic afterintravenous administration in mice, formal single dose toxicity testingwas performed over the course of 2 weeks using ¹²⁷I-RGD-PEG dots atabout 100 times of the human dose equivalent. ¹²⁷I-PEG dots served asthe control particle. In summary, the procedure was as follows.Twenty-eight, 8 week old B6D2F1 mice were used in the acute toxicitystudy and were divided into a treatment and control group. The treatmentgroup (n=6 males+6 females) received one dose ¹²⁷I-PEGylated RGD silicananoparticles at a dose of 1×10⁻⁹ moles/mouse intravenously, and thecontrol group (n=6 males+6 females) received the same amount of vehicle.Two mice/group (one male and one female/group) were sacrificed on day 7post dose and clinical chemistry, hematology and tissue specifichistopathology were done at autopsy. All remaining animals (n=5 males+5females/group) were observed for 14 days following treatment. Fouruntreated mice (two males and two females) were used as reference. Theconclusion of the studies was that no adverse events were observedduring dosing or the following 14-days observation period. No mortalityor morbidity was observed. Clinical observations included the absence ofthe following: anemia, weight loss, agitation, increased respiration, GIdisturbance, abnormal behavior, neurological dysfunction, abnormalitiesin hematology, abnormalities in clinical chemistries, or drug-relatedlesions in terms of organ pathology. Thus, a single injection of¹²⁷I-PEGylated RGD silica nanoparticles at 1×10⁻⁹ moles/mouse, a doseequivalent to an excess of 100 times the PEGylated RGD silicananoparticles dose required for Phase 0 imaging studies, is safe andnontoxic in B6D2F1 mice.

Efficient renal excretion was found for the ˜7-nm diameter targeted andnon-targeted probes over a 168-hr time period by fluorometric analysesof urine samples. Fluorescence signals were background-corrected andconverted to particle concentrations (% ID/μl) based on a serialdilution calibration scheme (FIG. 9C, inset; Table 4, column 2). Burns,et al., Fluorescent Silica Nanoparticles with Efficient UrinaryExcretion for Nanomedicine, Nano Letters, 9, 442-8 (2009). Concentrationvalues, along with age-dependent conservative estimates of the averageurine excretion rate, permitted the cumulative % ID excreted to becomputed (Table 4, column 4). Drickamer, Rates of urine excretion byhouse mouse (mus domesticus): differences by age, sex, social status,and reproductive condition. J. Chem. Ecol. 21, 1481-1493 (1995). Nearlyhalf of the injected dose (about 43% ID) was observed to be excretedover the first 24 hrs p.i. and ˜72% ID by 96 hrs, FIG. 9C), suggestingthat the bulk of excretion has occurred in the first day p.i. Nosignificant particle fluorescence in urine could be detected 168 hrsp.i. Fecal excretion profiles of the ¹²⁴I-cRGDY-PEG-dot indicated that,on average, 7% ID and 15% ID of the injected dose was eliminated over 24and 96 hrs, respectively (FIG. 9D). FCS analysis of urine samplesobtained at multiple time points after injection of the targeted proberevealed that the particle was excreted intact and without release ofthe encapsulated dye (data not shown).

TABLE 4 Urine Concentration and Cumulative Excretion Data Avg. UrineComputed Time Concentration Volume Cumulative % ID (hr) (% ID/ul) (μl)Excreted 7.0 nm 0 0.0 0.0 0 RGDY-PEG 1 0.292 41.6 6.07 dot 4 0.026 166.726.1 24 0.016 1000. 43.4 96 0.004 3974. 72.2

Serial Whole Body PET Studies

PET imaging of integrin expression in M21 and M21L subcutaneous hindlegxenograft mouse models was performed at multiple time points p.i.following i.v. injection of ¹²⁴ I-cRGDY-PEG-dots or ¹²⁴I-PEG-dots(control). Representative whole-body coronal microPET images at 4 hrs(left: M21 tumor; middle: M21L tumor) and 24 hrs (right: M21 tumor) p.i.are shown in FIG. 11A. The specific targeting of the α_(v)β₃integrin-overexpressing M21 tumor is clearly visible from these images.Average tumor % ID/g and standard deviations are shown for groups of M21(n=7) and M21L (control) tumors (n=5) receiving the targeted¹²⁴I-cRGDY-PEG-dots, as well as for M21 tumor mice (n=5) receivingnon-targeted ¹²⁴I-PEG-dot tracer (FIG. 11B). At the time of maximumtumor uptake (˜4 hrs p.i.), three-fold activity-concentration increases(in % ID/g) were seen in the M21 tumors over the controls. Differenceswere statistically significant at all time points p.i. (p<0.05) exceptat 1 hr (p=0.27).

Image-derived tumor-to-muscle uptake (% ID/g) ratios for the¹²⁴I-cRGDY-PEG-dots revealed enhanced tumor contrast at later times(˜24-72 hrs p.i.), while that for ¹²⁴I-PEG-dots declined (FIG. 11C).This finding suggested that ¹²⁴I-cRGDY-PEG-dots were, in fact,tumor-selective, which became more apparent as the blood activity wascleared during the initial 24-hr period (compare FIG. 11C with inset ofFIG. 9A). A statistically significant correlation was found betweenPET-derived tumor tissue % ID/g values for both ¹²⁴I-cRGDY-PEG-dots and¹²⁴I-PEGdots, and the corresponding ex-vivo γ-counted tumor % ID/gvalues (correlation coefficient r=0.94, P<0.0016; FIG. 11D), confirmingthe accuracy of PET for non-invasively deriving quantitativebiodistribution data.

In Vivo NIR Fluorescence Imaging and Microscopy

We performed in vivo fluorescence imaging studies using our small,targeted nanoparticles for mapping local/regional nodes and lymphaticchannels, thus overcoming the foregoing limitation. Importantly, themultimodal nature and small size of our targeted particle probe can beexploited to visualize a range of nodal sizes and lymphatic branches inour melanoma model following 4-quadrant, peritumoral administration,simulating intraoperative human sentinel lymph node mapping procedures.Initially, serial NIR fluorescence microscopy was performed in intactmice over a 4-hr time period using either the targeted or non-targetedparticle probes. Peritumoral administration of the targeted proberevealed drainage into and persistent visualization of adjacent inguinaland popliteal nodes over this interval, with smaller and/or more distantnodes and lymphatics more difficult to visualize. By contrast, thenon-targeted probe yielded shorter-term (˜1 hr) visualization of localnodes with progressively weaker fluorescence signal observed (data notshown). Upon surgical exposure, this observation was found to be theresult of more rapid particle diffusion from the tumor site, as comparedwith the extended retention observed with the targeted probe.

We next performed representative lymph node mapping over multiplespatial scales using live-animal whole-body optical imaging (FIG. 12A)and NIR fluorescence microscopy techniques (FIG. 12B) to visualizelymphatic drainage from the peritumoral region to the inguinal andaxillary nodes in surgically exposed living animals. In addition,higher-resolution fluorescence images (FIG. 12B, lower row) permittedmore detailed intranodal architecture to be visualized, including highendothelial venules, which facilitate passage of circulating naïvelymphocytes into the node, and which may have important implications fornodal staging and the ability to detect micrometastases at earlierstages of disease. Smaller, less intense lymphatic branches were alsovisualized by fluorescence microscopy in the axillary region (data notshown). Thus, the small size of the targeted probe not only permits thefirst draining (or sentinel node), proximal to the tumor to bevisualized, but also enables visualization of more distant nodes and ofthe pattern of lymphatic drainage to be visualized.

Discussion

We report on non-toxic, high-affinity, and efficiently cleared silicananoparticles for tumor-selective targeting and nodal mapping, havingsuccessfully addressed a number of the current challenges associatedwith other particle technologies. This is the first targetednanoparticle that, on the basis of its favorable properties, can be saidto be clinically translatable as a combined optical-PET probe. Thecomplementary nature of this multimodal probe, coupled with its smallsize (˜7-nm diameter), may facilitate clinical assessment by enablingthe seamless integration of imaging data acquired at different spatial,temporal, and sensitivity scales, potentially providing new insightsinto fundamental molecular processes governing tumor biology.

Our in vitro results show receptor-binding specificity of the ˜7-nmtargeted particle probe to M21 and HUVEC cells. Similar findings havebeen reported with receptor-binding assays using the same cell types,but with the monovalent form of the peptide. Cressman, et al., Bindingand uptake of RGD-containing ligands to cellular α_(v)β₃ integrins. IntJ Pept Res Ther. 15, 49-59 (2009). Importantly, the multivalencyenhancement of the cRGDY-bound particle probe, along with the extendedblood and tumor residence time T_(1/2) values, are key propertiesassociated with the particle platform that are not found with themonovalent form of the peptide.

The relatively long blood T_(1/2) value of 7.3±1.2 hrs estimated for the¹²⁴I-PEG-dot tracer may be related to the chemically neutral PEG-coatedsurface, rendering the probe biologically inert and significantly lesssusceptible to phagocytosis by the reticuloendothelial system. That areduction in the T_(1/2) value to 5.6±0.15 hrs was found for the¹²⁴I-cRGDY-PEG-dot tracer is most likely the result of recognition bytarget integrins and/or more active macrophage activity. However, it issubstantially longer than published blood T_(1/2) values of existingcRGDY peptide tracers (˜13 minutes), and results in greater probebioavailability, facilitating tumor targeting and yielding higher tumoruptakes over longer periods of time. Montet, et al., Multivalent effectsof RGD peptides obtained by nanoparticle display. J Med Chem. 49,6087-6093 (2006). In addition, the tumor T_(1/2) value for the¹²⁴I-cRGDY-PEG-dot was about 13 times greater than that for blood,versus only a fivefold difference for the ¹²⁴I-PEG-dot, suggestingsubstantially greater target-tissue localization of the former than thelatter. Such mechanistic interpretations of the in vivo data can beexploited to refine clinical diagnostic, treatment planning, andtreatment monitoring protocols.

The results of this study underscore the clear-cut advantages offered byPET, a powerful, quantitative, and highly sensitive imaging tool fornon-invasively extracting molecular information related to receptorexpression levels, binding affinity, and specificity. The greateraccumulation in and slower clearance from M21 tumors, relative tosurrounding normal structures, allows discrimination of specific tumoruptake mechanisms from non-specific mechanisms (i.e., tissue perfusion,leakage) in normal tissues. A small component of the M21 tumor uptake,however, presumably can be attributed to vascular permeabilityalterations (i.e., enhanced permeability and retention effects).Seymour, Passive tumor targeting of soluble macromolecules and drugconjugates. Crit. Rev. Ther. Drug Carrier Syst. 9, 135-187 (1992). Thisnon-specific mode of uptake reflects a relatively small portion of theoverall tumor uptake at earlier p.i. time points based on the observed %ID/g increases in mice receiving the control tracer (¹²⁴I-PEG-dots, FIG.11B). At 1-hr p.i., no significant % ID/g increases were seen in the M21tumors over the controls. This observation may reflect the effects ofdifferential perfusion in the first hour, with tumor accumulation andretention primarily seen at later p.i. times (i.e., 24 hrs). Further, incomparison with the clinically approved peptide tracer, ¹⁸F-galacto RGD,nearly two-fold greater uptake in M21 tumors was found for the¹²⁴I-cRGDY-PEG-dots34, while additionally offering advantages ofmultivalent binding, extended blood circulation times, and greater renalclearance.

One advantage of a combined optical-PET probe is the ability to assessanatomic structures having sizes at or well below the resolution limitof the PET scanner (i.e., the so-called partial-volume effect), whichmay undermine detection and quantitation of activity in lesions. Forinstance, in small-animal models, assessment of metastatic disease insmall local/regional nodes, important clinically for melanoma stagingand treatment, may not be adequately resolved by PET imaging, given thatthe size of the nodes observed are typically on the order of systemspatial resolution (1-2 mm). By utilizing a second complementary andsensitive imaging modality, near-infrared (NIR) fluorescence imaging,functional maps revealing nodal disease and lymphatic drainage patternscan be obtained. Ballou, et al., Sentinel lymph node imaging usingquantum dots in mouse tumor models. Bioconjugate Chem. 18, 389-396(2007). While further studies investigating the distribution ofintranodal cRGDY-PEG-dot fluorescence in relation to metastatic foci areneeded to determine whether sensitive localization of such foci can beachieved, these results clearly demonstrate the advantages of workingwith such a combined optical-PET probe.

In the clinic, the benefits of such a combined platform for tumorstaging and treatment cannot be overstated. The extended bloodcirculation time and resulting bioavailability of this nanoprobehighlights its use as a versatile tool for both early and long-termmonitoring of the various stages of disease management (diagnosticscreening, pre-treatment evaluation, therapeutic intervention, andpost-treatment monitoring) without restrictions imposed by toxicityconsiderations. An additional important advantage is that while rapidlycleared probes may be useful for certain applications where targettissue localization is itself rapid, localization of many agents inoften poorly vascularized and otherwise relatively inaccessible solidtumors will likely be slow following systemic administration. Thus, thecurrent nanoparticle platform expands the range of applications of suchagents, as the kinetics of target tissue localization are no longerlimiting. Furthermore, deep nodes can be mapped by PET in terms of theirdistribution and number while more precise and detailed localization ofsuperficial nodes can be obtained by NIR fluorescence imaging. Finally,the relatively prolonged residence of the targeted probe from tumorrelative to that from blood, in addition to its multivalencyenhancement, may be exploited for future theranostic applications as aradiotherapeutic or drug delivery vehicle.

Example 5 Fluorescent Silica Nanoparticles Conjugated with α_(v)β₃Integrin-Targeting Peptide and/or uMUC1-Targeting Peptide (ThyroidCancer and Squamous Cell Carcinoma (SCC) Models)

A cRGD peptide (Peptides International), having a cysteine endfunctionality, will be attached to the PEG-ylated nanoparticle via athiol-maleimide linkage. The nanoparticles can optionally further befunctionalized by a synthetic peptide ligand, EPPT1. The nanoparticleswill be characterized on the basis of particle size, size distribution,and photobleaching.

Characterization of Nanoparticle-Peptide Conjugates

For assessing photophysical properties on a per-particle basis,spectrophotometry, spectrofluorometry, and multiphoton fluorescencecorrelation spectroscopy (FCS) will be used to determine the particlesize, brightness, and size distribution. Size data will be corroboratedby scanning electron microscopy and dynamic light scattering (DLS)measurements. Ow et al. Bright and stable core-shell fluorescent silicananoparticles. Nano Letters 2005; 5, 113. Average number of RGD peptidesper nanoparticle and coupling efficiency of RGD to functionalized PEGgroups will be assessed colorimetrically under alkaline conditions andBiuret spectrophotometric methods (λ=450 nm, maximum absorbance).

The nanoparticle conjugates will be iodinated via tyrosine linkers tocreate a radiolabeled (¹²⁴I) (T_(1/2) ˜4 d) and stable (¹²⁷I) form byusing Iodogen51 (Pierce, Rockford, Ill.). The end product will bepurified by using size exclusion chromatography.

Evaluation of In Vitro Targeting Specificity and BiodistributionPatterns of the RGD- and RGD-EPPT-Nanoparticles.

α_(v)β₃ integrin and uMUC1 expression patterns in thyroid and squamouscell carcinoma (SCC) cell lines will be evaluated against known α_(v)β₃integrin-negative and α_(v)β₃ integrin-positive (M21-L and M21 humanmelanoma cell lines, respectively) and uMUC1-negative and uMUC1-positive(U87²⁸, H-29 cell lines, respectively) controls using anti-integrin andanti-uMUC1 antibodies. Cell lines highly expressing α_(v)β₃-integrinand/or MUC1 will be selected for differential binding studies with RGD-and RGD-EPPT-nanoparticles, as well as for in vivo imaging.

Quantitative cell binding assays will assess the labeling efficiency oftumor cells, and biodistribution studies assaying uptake in tumor,organs, and fluids will be performed using radioiodinated nanoparticleconjugates (¹²⁴I-RGD-nanoparticles, ¹²⁴I-RGD-EPPT-nanoparticles). Tocompare PET uptake data of nanoparticle conjugates with that observedinitially using optical NIRF imaging, each nanoparticle conjugate willalso be iodinated to create a radiolabeled (¹²⁴I) and stable (¹²⁷I)form.

Fluorescence Microscopy with RGD- and RGD-EPPT-C-dots. Differentialbinding of RGD-nanoparticles and RGD-EPPT-nanoparticles to thyroidcarcinoma/SCC cell lines highly expressing α_(v)β₃-integrin and/or MUC1,versus control lines will be visualized by fluorescence microscopy.

Animal models. All animal experiments will be done in accordance withprotocols approved by the Institutional Animal Care and Use Committeeand following NIH guidelines for animal welfare.

In vivo Biodistribution: Male athymic nude mice (6-8 week old, n=5 pertumor) will be subcutaneously (s.c.) injected in both flanks withintegrin-negative/-positive or uMUC1-negative/-positive tumors ofdifferent tissue origins (n=3/each tumor). At 0.5 cm in diameter (i.d.),mice will be injected intravenously (IV) with ¹²⁴I-labeled nanoparticleconjugates (˜500 nm/kg). Animals are sacrificed at 0.5, 1, and 24-hrslater, with removal of tumors, organs, and fluids for weighing andcounting (gamma counter). Biodistribution results will be expressed asthe percentage of injected dose per gram of tissue.

Quantitative Cell Binding Assay. Labeling efficiency will be assessed byincubating fixed numbers of carcinoma cells highly expressingα_(v)β₃-integrin and/or MUC1, with pre-selected concentrations of¹²⁴I-labeled nanoparticle conjugates for 1-hr in a humidified CO₂atmosphere at 37° C. Cells are extensively washed, lysed with 0.1%Triton X, with cell lysates counted in a gamma counter.

Assess of Relative Differences in Tumor-Specific Targeting Using In VivoMultimodality (PET-NIRF) Imaging.

As a high-throughput diagnostic screening tool, optical NIRF imaging canbe used to evaluate relative differences in the biodistribution ofprogressively functionalized nanoparticle conjugates in vivo withincreased sensitivity and temporal resolution. Semi-quantitative data ontumor-specific targeting can also be derived. These preliminary studiesfacilitate the selection of cell lines strongly expressing markers ofinterest for further detailed quantitation of biodistribution andtumor-specific targeting using PET.

Whole-body microPET™ and NIRF optical imaging will be performed over a1-week period to assess differential uptake in flank tumors. The resultsof these studies will be validated with fluorescence microscopy oftumors ex-vivo.

Serial In Vivo NIRF Imaging. Mice will be injected bilaterally withα_(v)β₃ integrin-negative and α_(v)β₃ integrin-positive cells or withuMUC1-negative and uMUC1-positive cells (n=5/tumor). After tumors reach˜0.5 cm i.d., stable iodinated and non-iodinated nanoparticle conjugates(RGD, ¹²⁷I-RGD, RDG-EPPT, ¹²⁷I-RGD-EPPT) will be injected IV. Serialimaging will be performed using the Maestro™ In Vivo FluorescenceImaging System (CRI, Woburn, Mass.) at 0, 0.5, 1, 2, 4, 6, 12, and 24hrs. At 24-h, mice are euthanized, and major tissues/organs dissected,weighed, and placed in 6-well plates for ex-vivo imaging. Fluorescenceemission will be analyzed using regions-of-interest (ROIs) over tumor,selected tissues, and reference injectates, employing spectral unmixingalgorithms to eliminate autofluorescence. Dividing average fluorescenceintensities of tissues by injectate values will permit comparisons to bemade among the various tissues/organs for each injected nanoparticleconjugate.

Dynamic MicroPET Imaging Acquisition and Analysis. Two groups oftumor-bearing mice (n=5/tumor) will be injected with radiolabeled¹²⁴I-nanoparticle conjugates (radiotracers), and dynamic PET imagingperformed for 1-hr using a Focus 120 microPET™ (Concorde Microsystems,TN). One-hour list-mode acquisitions are initiated at the time of IVinjection of ˜25.9 MBq (700 μCi) radiotracers. Resulting list-mode dataare reconstructed in a 128×128×96 matrix by filtered back-projection.ROI analysis of reconstructed images is performed using ASIPro™ software(Concorde Microsystems, TN) to determine the mean and SD of radiotraceruptake (% ID/g) in tumors, other organs/tissues, and left ventricle(LV). Additional data will be obtained from static images at 24-, 48-,and 72-hr post-injection time points. A three-compartment,four-parameter kinetic model will be used to characterize tracerbehavior in vivo. For this analysis, arterial input is measured using anROI placed over the LV.

Example 6—Nodal Mapping in Miniswine

Real-time intraoperative scanning of the nodal basin cannot bepractically achieved at the present time, as these systems are generallytoo cumbersome and expensive for use in the operating suite or may beunable to provide the necessary field-of-view or tissue contrast.Further, there are no clinically promising, biostablefluorophore-containing agents, offering improved photophysical featuresand longer circulation lifetimes over parent dyes, available to enhancetissue contrast for extended nodal mapping/resection procedures. Withthis animal study, we will show that advances in both multimodalparticle probes and real-time molecular imaging device technologies canbe readily translated to a variety of future human clinical trials. Suchtransformative technologies can significantly impact standardintraoperative cancer care by providing state-of-the-art targetedvisualization tools for facilitating metastatic SLN detection andenabling accurate delineation of node(s) from adjoining anatomy tominimize risk of injury to crucial structures. Benefits include extendedreal-time in vivo intraoperative mapping of nodal disease spread andtumor extent in the head and neck. Deep nodes can be mapped by PET,while precise and detailed localization of superficial nodes can beobtained by NIR fluorescence imaging. The small size of the particleprobe may also extend the lower limit of nodal sizes that can besensitively detected. The net effect of the proposed non-toxic,multimodal platform, along with the application of combineddiagnostic/treatment procedures, has important implications for diseasestaging, prognosis, and clinical outcome for this highly lethal disease.

Disease Target.

In addition to melanoma, a number of other tumors (i.e., breast, lung,and brain) overexpress αvβ3 integrin receptors and could serve asdisease targets. Metastatic melanoma has a very poor prognosis, with amedian survival of less than 1 year 11. Successful management relies onearly identification with adequate surgical excision of the cancer.Surgical removal of the primary disease, screening, and treatment forregional lymph node spread is standard-of-care in the US to accuratelystage disease and tailor treatment. The recently revised stagingguidelines recognize the presence of microscopic nodal metastases as ahallmark of advanced stage disease leading to dramatically reducedsurvival. Knowledge of pathologic nodal status is critical for earlyrisk stratification, improved outcome predictions, and selection ofpatient subgroups likely to benefit from adjuvant treatment (therapeuticnodal dissection, chemotherapy) or clinical trials.

Sentinel Lymph Node (SLN) Mapping.

SLN mapping techniques, routinely used in staging melanoma, identify thespecific node(s) that are at highest risk of tumor metastases. Thisprocedure identifies patients harboring metastatic disease for furthertreatment. Standard-of-care techniques rely on injection of radioactivetechnetium (^(99m)Tc) sulfur colloid dye around the primary tumor forSLN localization, followed by the intraoperative use of a gamma probe tomeasure radioactivity in lymphatic structures within an exposed nodalbasin. Blue dye injected about the primary tumor can help delineatesmall SLN(s) from adjacent tissue, but the technique is unreliable andliable to complications. Current SLN mapping and biopsy techniques havelimitations, and account for higher rates of non-localization of SLN(s)in the head and neck compared to other anatomic sites. The head and neckregion is notorious for its unpredictable patterns of metastaticdisease. The close proximity of the primary disease to nodal metastasesin this region makes intraoperative use of the gamma probe difficult dueto interference from the injection site. Importantly, current technologydoes not allow the surgeon to visualize the sentinel node and reliablydifferentiate it from adjoining fat or other tissues, placing vitalstructures (i.e., nerves) at risk for injury during dissection toidentify and harvest this node. The small size of nodes and widevariation in drainage patterns provides additional challenges, resultingin a non-localization rate of around 10%.

Nanoparticles.

The majority of preclinical studies have used RGD peptide orpeptide-conjugate radiotracers as targeting ligands for imagingαvβ3-integrin expression. ¹⁸F-galacto-RGD and ^(99m)Tc-NC100692 arepeptide tracers that have been used successfully in patients to diagnosedisease. Peptide tracers clear rapidly, which may result in reducedreceptor binding and increased background signal from non-specifictissue dispersal. These properties limit the potential of peptidetracers for longer-term monitoring. By contrast, nanoparticle probes(˜10-100 nm), which have also been used for imaging integrin expressionalong tumor neovasculature, have extended circulation half times forperforming longer-term monitoring (i.e., days). Nanoparticles aretypically larger than antibodies and radiopharmaceuticals (<10 kDa), andare associated with slower transmembrane transport, increased RESuptake, and enhanced non-specific uptake due to altered tumor vascularpermeability. The 7 nm diameter targeted nanoparticles used for this SLNmapping study are roughly comparable to the average diameter of analbumin molecule and 2-3 times smaller than the average diameter of atypical antibody. Relative to peptide tracers, the targeted particleprobe is less prone to extravasation and is associated with extendedcirculation half times that enhance tumor targeting. Importantly,124I-cRGDY-PEG-dots demonstrate key in vitro and in vivo properties inM21 tumors necessary for clinical translation.

Materials and Methods.

Spontaneous melanoma Sinclair miniature swine (10-12 kg, SinclairResearch Center, MO) were injected intravenously with 5 mCi¹⁸F-fluoro-deoxyglucose (¹⁸F-FDG) for whole-body screening of nodaland/or organ metastases. Miniswine underwent 1-hr dynamic ¹⁸F-FDG PETwhole body PET scan using a clinical PET scanner 40 minutes afterinjection to screen for metastatic disease, followed by CT scanacquisition for anatomic localization. Then miniswine were subdermallyinjected in a 4-quadrant pattern about the tumor site (head and necksites preferentially) with multimodal ¹²⁴I-RGD-PEG-dots 48 hrs after¹⁸F-FDG PET, and a second dynamic PET-CT scan performed to assess foradditional nodal metastases.

Miniswine were taken to the operating room for identification of nodes.Optical fluorescence imaging was performed using large field-of-viewnear infrared fluorescence camera system, smaller field-of-view modifiedendoscope, and a modified stereomacroscope for obtaining higherresolution fluorescence images within the exposed surgical bed.

Validation of the fluorescent signal was performed intraoperatively bygamma counting with a clinically-approved hand-held PET device withinthe operative bed to localize targeted dots transdermally, acquiredintraoperatively from skin and the nodes within and nodal basin.

The primary melanoma skin lesion was excised, and an incision made toallow access to the sentinel node(s). Nodal identity was confirmed usinghand held PET and multi-scale optical imaging systems, and the nodes inquestion excised. Specimens was sent for histological assessment formetastases and optical confocal microscopy to confirm the presence ofboth malignancy and nanoparticle fluorescence.

Following harvest of the sentinel nodes, the entire lymph node basin wasexcised and further evaluated using histological methods (withimmunohistochemical markers for melanoma as needed), fluorescencemicroscopy, and the hand-held PET probe for correlative purposes. Thisstep helped identify any other malignant nodes within the nodal basinand the number of ¹²⁴I-RGD-PEG-dots present in adjacent nodes by theirappearance on imaging.

¹²⁴I-RGD-PEG-dots was administered subcutaneously into the limbs of theanimal sequentially. Transit of the ¹²⁴I-RGD-PEG-dots to theinguinal/axillary nodes was followed using the optical imaging systemand hand held PET probes to confirm the duration of transit along thelymphatic pathways. The draining nodal basins was exposed surgically andthe pattern of lymph node drainage observed. The sentinel lymph node washarvested from each site to confirm the lymphatic nature of the tissue.Animals were euthanized, and any further lesions noted on imaging wereexcised in the necropsy room of the animal facility.

Discussion.

A whole-body ¹⁸F-fluorodeoxyglucose (¹⁸F-FDG) PET-CT scan revealed aprimary melanomatous lesion adjacent to the spine on the upper back, aswell as a single node in the neck, posteriorly on the right side of theanimal, which were both FDG-avid, and suspicious for metastatic disease.This finding was confirmed after subdermal, 4-quadrant injection of¹²⁴I-RGD-PEG-dots about the tumor site, which additionally identifiedtwo more hypermetabolic nodes, as well as the draining lymphatics. Finalscan interpretation pointed to 3 potential metastatic nodes. Surgicalexcision of the primary lesion, hypermetabolic nodes, and tissue fromother nodal basins in the neck bilaterally was performed after hand-heldPET probes identified and confirmed elevated count rates at the locationof sentinel node(s). Patchy fluorescence signal measured in the excisedright posterior sentinel node tissue correlated with sites of melanomametastases by histologic analysis. All hypermetabolic nodal specimenswere black-pigmented, and found to correlate with the presence ofdistinct clusters of melanoma cells. Thus, the results of surgicallyresected tissue submitted to pathology for H&E and staining for otherknown melanoma markers confirmed multimodal imaging findings.

FIG. 13A shows the experimental setup of using spontaneous miniswinemelanoma model for mapping lymph node basins and regional lymphaticsdraining the site of a known primary melanoma tumor. This intermediatesize miniswine model is needed to simulate the application of sentinellymph node (SLN) biopsy procedures in humans, and more accuratelyrecapitulates human disease. FIG. 13B shows small field-of-view PETimage 5 minutes after subdermal injection of multimodal particles(¹²⁴I-RGD-PEG-dots) about the tumor site. The tumor region, lymph nodes,and the lymphatics draining the tumor site are seen as areas ofincreased activity (black).

FIGS. 14A-14C show whole-body dynamic ¹⁸F-fluorodeoxyglucose (¹⁸F-FDG)PET scan (FIG. 14A) and fused ¹⁸F-FDG PET-CT scans (FIG. 14B)demonstrating sagittal, coronal, and axial images through the site ofnodal disease in the neck. The ¹⁸F-FDG PET scan was performed to mapsites of metastatic disease after intravenous administration and priorto administration of the radiolabeled nanoparticle probe. A singlehypermetabolic node is seen in the neck posteriorly on the right side ofthe animal (arrows, axial images, upper/lower panels), also identifiedon the whole body miniswine image (FIG. 14C).

FIGS. 15A-15C show the same image sets as in FIGS. 14A-14C, but at thelevel of the primary melanoma lesion, adjacent to the spine on the upperback. The PET-avid lesion is identified (arrows, axial images,upper/lower panels), as well as on the whole body miniswine image (FIG.15C).

FIGS. 16A-16C show high resolution dynamic PET (FIG. 16A) and fusedPET-CT images (FIG. 16B) following subdermal, 4-quadrant injection of¹²⁴I-RGD-PEG-dots about the tumor site, simulating clinical protocol,over a 1 hour time period. Three hypermetabolic lymph nodes (arrows)were found in the neck, suggesting metastatic disease. The excised rightposterior SLN was excised and whole body near infrared (NIR)fluorescence imaging was performed. Cy5 fluorescence signal wasdetectable within the resected node (FIG. 16C, top, Cy5 imaging) onwhole-body optical imaging. Pathological analysis of thisblack-pigmented node (arrow, SLN) demonstrated clusters of invadingmelanoma cells on low-(arrows) and high-power cross-sectional views ofthe node by H&E staining (lower two images), and we expect melanomaspecificity to be further confirmed using special stains (Melan A,HMB45, PNL2, and “melanoma associated antigen” biogenex clone NKI/C3).We additionally expect colocalization of the particle with thesemetastatic clusters of cells on confocal fluorescence microscopy andhigh resolution digital autoradiography, confirming metastatic diseasedetection.

Example 7 Fluorescent Silica Nanoparticles Conjugated withMC1R-Targeting Peptide (Melanoma Model)

For the multimodality (PET-NIRF) imaging experiments, the targetingpeptide and the radiolabel on the nanoparticle surface will be exchangedto determine target specificity, binding affinity/avidity, and detectionsensitivity. Nanoparticles will be synthesized using therapeuticradiolabels (lutetium-177, ¹⁷⁷Lu, t^(1/2)=6.65 d) for targeted killingof MC1R-expressing melanoma cells. Combined quantitative PET and opticalimaging findings will be correlated with tumor tissue autoradiographyand optical imaging across spatial scales. For cellular microscopy, anin vivo confocal fluorescence scanner for combined reflectance andfluorescence imaging will be used.

Example 8 Fluorescent Nanoparticles for Targeted Radiotherapy

Dose escalation studies with ¹³¹I-RGD nanoparticles will be performedand treatment response will be monitored weekly, over the course of sixweeks, using ¹⁸F-FDG PET. Time-dependent tumor uptake and dosimetry ofthe nanoparticle platform will be performed using planar gamma cameraimaging. In vivo imaging data will be correlated with gamma counting ofexcised tumor specimens.

Male nude mice (6-8 wks, Charles River Labs, MA) will be used forgenerating hind leg xenograft models after injection of M21 humanmelanoma cells (5×10⁵ in PBS). Tumors will be allowed to grow 10-14 daysuntil 0.5-0.9 cm³ in size.

¹³¹I-based targeted radiotherapy studies. The therapeutic radionuclide¹³¹ I will be used as a radiolabel for targeted radiotherapy. Inestimating the highest possible ¹³¹I dose resulting in no animal deathsand less than 20% weight loss (MTD), a dose escalation study will becarried out in tumor-bearing nude mice. For a 200 rad dose to blood 54,an administered activity of 10 MBq is required, which would deliver adose of 270 rad to tumor. 4 doses of 10 MBq each will be administered toachieve a tumor dose greater than 1000 rad with dose fractionationdesigned to allow repair and sparing of bone marrow. ¹³¹I allows forplanar gamma camera imaging using a pinhole collimator to measure thetime-dependent tumor uptake and dosimetry of the nanoparticles. ¹⁸F-FDGPET allows for quantitative monitoring of tumor response, thus providingcomplementary information.

Based on this data, and in vivo data on the effect of nanoparticlesloaded with paclitaxel, a therapy study with the ¹³¹I -RGD-nanoparticleconjugate will be conducted. Two groups of tumor-bearing mice (n=10 pergroup) will receive either four, 10.4-MBq activities once per week for 4weeks, of i.v.-administered ¹³¹I -RGD-nanoparticle conjugates or salinevehicle (control, n=10), and will be monitored over a 6-week period.Treatment response/progression will be quantified on the basis of tumorvolume (via caliper measurements). All mice from the treatment groupswill also be imaged once per week (˜1 hr sessions) by SPECT imaging(Gamma Medica) over a 6 week period.

¹⁸F-FDG PET Imaging Acquisition and Analysis. Two groups oftumor-bearing mice (n=10/group) will undergo initial PET scanning priorto and then, on a weekly basis after treatment over a 6 week interval.Mice will be injected intravenously (i.v.) with 500 μCi ¹⁸F-FDG andstatic 10-minute PET images will be acquired using a Focus 120 microPET™(Concorde Microsystems, TN) before and after treatment. Acquired datawill be reconstructed in a 128×128×96 matrix by filteredback-projection. Region-of-interest (ROI) analyses of reconstructedimages will be performed using ASIPro™ software (Concorde Microsystems,TN) to determine the mean and SD of radiotracer uptake (% ID/g) intumors. Animals will be sacrificed at the termination of the study andtumors excised for gamma counting.

Example 9 Fluorescent Nanoparticles Conjugated with Radionuclide Chelateand MC1R-Targeting Peptide

PEG-ylated nanoparticles will be conjugated with targeting peptides andmacrocyclic chelates binding high-specific activity radiolabels.

High purity two-arm activated commercially available PEGs, derivatizedwith NHS esters or maleimide, will be attached to the silica shell ofthe nanoparticle using standard procedures. Either of the twofunctionalized PEG groups (NHS esters or maleimide) will be availablefor further conjugation with either the peptide-chelate construct,cyclic peptide Re-[Cys3,4,10,D-Phe7]α-MSH3-13 (ReCCMSH(Arg11)), or1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid (DOTA)linker chelators. The covalent attachment of derivatized PEGs to thenanoparticle surface will be performed in such a manner as to exposedifferent functional groups for linking DOTA and peptide-chelateconstructs, as discussed below.

Synthesis and Physicochemical Characterization of FunctionalizedNanoparticles.

Functionalized nanoparticles will be synthesized by establishingcovalent linkages of the following moieties with the derivatized PEGgroups:

(A) DOTA chelates for subsequent high-specific activity radiolabelingwith positron-emitting radiometals (i.e., ⁶⁴Cu) to permit diagnosticdetection with PET imaging. DOTA will be conjugated to thefunctionalized PEGs using standard Fmoc chemistry, and purification ofthe chelated nanoparticles will be performed by chromatography. ⁶⁴Cu and¹⁷⁷Lu will be attached to DOTA by incubation of the reaction mixture at60° C. for 30 min followed by gel filtration or high pressure liquidchromatography purification. Alternatively, PET nuclides, such as ¹²⁴I,⁸⁶Y, ⁶⁸Ga and ⁸⁹Zr, may be conjugated to the nanoparticle, either viathe DOTA-functionalized PEG (radiometals) or tyrosine-functionalized PEG(¹²⁴I) The single photon emitter, ¹⁷⁷Lu, obtained in the form of¹⁷⁷LuCl₃ will be complexed to DOTA for radiotherapy.

(B) αMSH melanoma targeting peptide analogue (ReCCMSH(Arg11)) iscyclized by rhenium. It is necessary to confirm the ratio of DOTAchelates to ReCCMSH(Arg11) moieties on the PEG-ylated nanoparticlesurface.

Characterization of the functionalized nanoparticle preparations will beperformed as follows:

(A) Average number of DOTA chelates per nanoparticle will be determinedby standard isotopic dilution assays with ⁶⁴Cu. Briefly, ⁶⁴Cu will beadded to solutions containing a known amount ofReCCMSH(Arg11)-nanoparticles. Incubated solutions will be spotted onsilica gel-coated glass plates, developed in 1:1 10% ammoniumacetate-to-methanol (with EDTA), and analyzed by radio-TLC. While⁶⁴Cu-labeled ReCCMSH(Arg11)-Nanoparticles will remain at the origin,⁶⁴Cu bound to EDTA will migrate. The percent labeling efficiency will beplotted against total nanomoles of ⁶⁴Cu added to the reaction mixture.The number of chelates attached per nanoparticle can be determined fromthe inflection point of this curve.

(B) Average number of ReCCMSH(Arg11) peptides per nanoparticle andcoupling efficiency of the ReCCMSH(Arg11) to the functionalized PEGgroups will be assessed using spectrophotometric methods (λ=435 nm,maximum absorbance) and the known extinction coefficient ofReCCMSH(Arg11). The incorporation of rhenium offers the advantage thathighly sensitive absorbance measurements of rhenium concentrations canbe made on a small sample of product.

In Vitro and In Vivo Optical-PET Imaging of Multifunctional NanoparticleNanoparticles in Melanoma Models to Assess Tumor-Specific Targeting andTreatment Response.

⁶⁴Cu-DOTA-ReCCMSH(Arg11)-nanoparticles will be compared with the native⁶⁴Cu-DOTA-ReCCMSH(Arg11) construct to test targeting capabilities of thenanoparticles.

Competitive binding assays. The MC1R receptor-positive B16/F1 murinemelanoma lines will be used. The IC₅₀ values of ReCCMSH(Arg11) peptide,the concentration of peptide required to inhibit 50% of radioligandbinding, will be determined using ¹²⁵I-(Tyr2)-NDP7, a radioiodinatedα-MSH analog with picomolar affinity for the MC1R. Single wells will beincubated at 25° C. for 3 h with approximately 50,000 cpm of¹²⁵I-(Tyr2)-NDP in 0.5 ml binding medium with 25 mmol/LN-(2-hydroxyethyl)-piperazine-N-(2-ethanesulfonic acid), 0.2% BSA and0.3 mmol/L 1,10-phenanthroline], with concentrations of (Arg11)CCMSHranging from 10-13 to 10-5 mol/L. Radioactivity in cells and media willbe separately collected and measured, and the data processed to computethe IC₅₀ value of the Re(Arg11)CCMSH peptide with the Kell softwarepackage (Biosoft, MO).

Receptor Quantitation Assay. Aliquots of 5×105 B16/F1 cells will beadded to wells, cultured in 200 μL RPMI media, and incubated at 37° C.for 1.5 h in the presence of increasing concentrations of¹²⁵I-(Tyr2)-NDP (from 2.5 to 100 nCi) in 0.5 mL of binding media (MEMwith 25 mM HEPES, pH 7.4). Cells will be washed with 0.5 mL of ice-cold,pH 7.4, 0.2% BSA/0.01 M PBS twice, and the level of activity associatedwith the cellular fraction measured in a γ-counter. Nonspecific bindingwill be determined by incubating cells and ¹²⁵I-(Tyr2)-NDP withnon-radioactive NDP at a final concentration of 10 μM. Scatchard plotswill be obtained by plotting the ratio of specific binding to free¹²⁵I-(Tyr2)-NDP vs. concentration of specific binding (fmol/millioncells); Bmax, the maximum number of binding sites, is the X intercept ofthe linear regression line.

B16/F1 murine melanoma lines (5×10⁵ in PBS) will be injectedsubcutaneously into the hind legs of Male nude mice (6-8 week old). Thetumors will be allowed to grow 10-14 days until 0.5-0.9 cm³ in size.

Biodistribution: A small amount of the⁶⁴Cu-DOTA-ReCCMSH(Arg11)-nanoparticle conjugate (˜10 μCi, 0.20 μg) willbe injected intravenously into each of the mice bearing palpable B16/F1tumors. The animals will be sacrificed at selected time points afterinjection (2, 4, 24, 48, 72 hours; n=4-5/time point) and desired tissuesremoved, weighed, and counted for accumulated radioactivity. Additionalmice (n=5) injected with the native radiolabeled construct,⁶⁴Cu-DOTA-ReCCMSH(Arg11) (˜10 μCi, 0.20 μg) will serve as the controlgroup, and evaluated 1 h post-injection. To examine in vivo uptakespecificity, an additional group of mice (2-h time point) will bepre-injected with 20 μg of NDP to act as a receptor block immediatelyprior to the injection of the ⁶⁴Cu-DOTA-ReCCMSH(Arg11) nanoparticleconjugate. Major organs and tissues will be weighed and gamma-counted,and the percentage-injected dose per gram (% ID/g) determined.

Serial In Vivo NIRF Imaging. In parallel with the PET studies below, NIR(fluorescence tomographic imaging, FMT 225, Visen, Woburn, Mass.) willbe performed using a tunable 680 nm scanning NIR laser beam and CCDbefore and after i.v. injection of tumor-bearing animals (n=10). Micewill be kept under continuous isoflurane anesthesia, and placed in aportable multimodal-imaging cassette (compatible with both our FMT 2500and Focus 120 microPET) for FMT scanning before and after injection (1,2, 4, 6, 12, 24, 48 and 72 hours). The NIR fluorescence image, measuredover a 1-10 minute period, will be reconstructed using the Visenproprietary software and superimposed onto a normal photograph of themouse. The imaging data is quantitative, as the measured intensity isdirectly related to the NIR fluorophore concentration, enablingparametric maps of absolute fluorophore concentrations to be generatedfor co-registeration with the acquired PET imaging data.

Dynamic PET Imaging Acquisition and Analysis. Two groups oftumor-bearing mice (n=5/group) will be placed in the imaging cassettefor co-registering sequential PET-optical studies. Mice will be injectedintravenously (i.v.); one with radiolabeled ⁶⁴Cu-DOTA-ReCCMSH(Arg11)nanoparticle conjugates and the second with native⁶⁴Cu-DOTA-ReCCMSH(Arg11) constructs. Following injection, dynamic 1-hrPET images will be acquired using a Focus 120 microPET™ (ConcordeMicrosystems, TN). One-hour list-mode acquisitions are initiated at thetime of IV injection of radiolabeled probe (˜1 mCi). Resulting list-modedata will be reconstructed in a 128×128×96 matrix by filteredback-projection. Region-of-interest (ROI) analyses of reconstructedimages are performed using ASIPro™ software (Concorde Microsystems, TN)to determine the mean and SD of radiotracer uptake (% ID/g) in tumors,other organs/tissues, and left ventricle (LV). Tracer kinetic modelingof the data will permit estimation of pharmacokinetic parameters,including delivery, clearance, and volume of distribution. As noted, anarterial blood input is measured using an ROI placed over the LV (as ameasure of blood activity). Additional data will be obtained from staticimages at 24 hr, 48 hr, 72 hr post-injection time points.

Fluorescence microscopy and autoradiography of tissues. A combination ofoptical imaging technologies exhibiting progressively smaller spatialscales (i.e., whole body fluorescence imaging, fluorescence macroscopy,and in vivo fluorescence confocal laser scanning microscopy) will beutilized for imaging tumors in live, intact animals at 72-hpost-injection. Mice will be maintained under continuous isofluoraneanesthesia, thus enabling detection and localization of fluorescencesignal from the whole animal/organ level to the cellular level over arange of magnifications. Whole animal/macroscopic imaging will beperformed with fluorescence stereomicroscope (Visen; Nikon SMZ1500)fitted with Cy5 fluorescence filter sets and CCD cameras. Fluorescenceconfocal laser scanning microscopy capabilities will be developed. Micewill subsequently be euthanized for autoradiography in order to maptracer biodistributions at high resolution throughout the tumor volume.Tumors will be excised, flashfrozen, serially sectioned (1□0μ sections)and slide-mounted, with alternating slices placed in contact with aphosphor plate in a light-tight cassette (up to 1 wk). H&E staining willbe performed on remaining consecutive sections. Autoradiographicfindings will be correlated with PET imaging data and histologicalresults.

The therapeutic radionuclides ¹⁷⁷Lu or ⁹⁰Y may alternatively be used fortargeted radiotherapy. In estimating the highest possible ¹⁷⁷Lu doseresulting in no animal deaths and less than 20% weight loss (MTD), adose escalation study will be carried out in tumor-bearing nude mice.Doses of radiopharmaceutical suspected to be at (or near) the MTD basedon literature values for ¹⁷⁷Lu will be evaluated.

Example 10 Fluorescent Nanoparticles Functionalized to Conjugate withLigand and Contrast Agent Via “Click Chemistry” Synthesis ofNanoparticles Containing Versatile Functional Groups for SubsequentConjugation of Ligand (e.g., Peptides) and Contrast Agent (e.g.,Radionuclides).

In order to synthesize an array of nanoparticle-peptide-chelateconstructs suitable for high-specific activity radiolabeling, a“click-chemistry” approach may be used to functionalize the nanoparticlesurface (FIG. 17). This method is based on the copper catalyzedcycloaddition of azide to a triple bond. Such an approach would allowfor a great deal of versatility to explore multimodality applications.

Nanoparticle synthesis and characterization. The PEG groups that will becovalently attached will be produced following the scheme in FIGS.14A-14C. PEG will be covalently attached to the nanoparticle via thesilane group. Standard chemical pathways will be used for the productionof the functionalized PEG with triple bonds.

Functionalization of nanoparticles with triple bonds. To synthesize thebi-functionalized PEGs, the first step will employ the well studiedreaction of activated carboxylic ester with aliphatic amine (FIG. 18).Alternatively, another suitable triple-bond bearing amine, for example,p-aminophenylacetylene, can be used. The second step of the synthesisalso relies on a well-known conjugation reaction.

Synthesis and Physicochemical Characterization of FunctionalizedNanoparticles Conjugated with Model Peptides and Chelates.

The functionalized nanoparticle contains both (A) desferrioxamine B(DFO) for subsequent high-specific activity radiolabeling with thepositron-emitter zirconium-89 (⁸⁹Zr) and (B) the SSTR-targeting peptide,octreotate.

Synthesis of DFO with an azide bond. DFO with an azide group will beproduced by reaction of DFO-B with pazido benzoic acid) (FIG. 19) andpurified. The “click chemistry” reaction is a 1,3-dipolar cycloadditionat room temperature and the conditions are often referred to as “HuigsenConditions”. Although the reactions can generally be completed at roomtemperature in ethanol, it may be appropriate to heat the reaction. Thecatalyst is often Cu(I)Br, but alternatives include Cu(I)I or Cu(II)SO4(with a reductant). Knor et al. Synthesis of novel1,4,7,10-tetraazacyclodecane-1,4,7,10-tetraacetic acid (DOTA)derivatives for chemoselective attachment to unprotectedpolyfunctionalized compounds. Chemistry, 2007; 13:6082-90. Clickreactions may also be run in the absence of any catalyst. Alternatively,the NH³⁺ group in DFO-B may be converted directly into an azide group.

Synthesis of Tyr3-octreotate with an azide. Solid phase peptidesynthesis (SPPS) of Tyr3-octreotate (FIG. 20A) will be performed on apeptide synthesizer. Briefly, the synthesis will involve the Fmoc(9-fluorenylmethoxycarbonyl) method as previous described for thispeptide. Briefly, the instrument protocol requires 25 μmol of subsequentFmoc-protected amino acids activated by a combination of1-hydroxybenzotriazole (HOBt) and2-(1Hbenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate(HBTU). The Fmoc-protected amino acids will be purchased commerciallyunless otherwise stated; the pre-packed amino acids will be obtainedfrom Perkin-Elmer (Norwalk, Conn.), while those unavailable inpre-packed form, such as the Damino acids and Fmoc-Cys(Acm) will besupplied by BACHEM Bioscience, Inc. (King of Prussia, Pa.) orNovabiochem (San Diego, Calif.). The azide group (for the “click”chemistry) will be introduced into the peptide backbone via coupling ofan azide-containing acid to the N-terminus of the peptide, while thepeptide is still protected and attached to the resin (FIG. 20B).

Synthesis of functionalized nanoparticles. The next step will be toconjugate both the DFO having an azide bond and Tyr3-octreotate havingan azide bond (FIGS. 21A and 21B) to the nanoparticle. “Click chemistry”is highly selective, quantitative and can be performed very fast andusing mild conditions. The number of combined azide groups from DFO andTyr3-octreotate will be controlled to never exceed the number ofavailable triple-bonds; the triple bonds will always be in <5% excess.

Functionalized nanoparticle characterization. Average number of DFOchelates peptide per nanoparticle will be determined by performing astandard isotopic dilution assay with ⁸⁹Zr (or ⁶⁸Ga). ⁸⁹Zr will beproduced on cyclotron and purified. Briefly, 10 concentrations of89Zr-oxalate will be added to solutions containing a known amount ofDFO-derived nanoparticles. Following a 30 min. room temperatureincubation, the solutions will be spotted on silica gel coated glassplates, developed in 1:1 10% ammonium acetate-to-methanol (with EDTA)and analyzed by radio-TLC. Whereas the ⁸⁹Zr -DFO-derived nanoparticleswill remain at the origin, nonspecifically bound ⁸⁹Zr bound to EDTA willmigrate. The percent labeling efficiency will be plotted as a functionof total nanomoles of ⁸⁹Zr added to the reaction mixture. The number ofchelates attached to the nanoparticle can then be determined from theinflection point of this curve.

Average number of Tyr3-octreotate peptide per nanoparticle will bedetermined by assaying the disulfide bridge of Tyr3-octreoate. Briefly,the disulfide bonds of the Tyr3-octreotate can be cleaved quantitativelyby excess sodium sulfite at pH 9.5 and room temperature. DTNB or Elman'sreagent can be used to quantitate thiols in proteins by absorptionmeasurements. It readily forms a mixed disulfide with thiols, liberatingthe chromophore 5-merapto-2-nitrobenzoic acid (absorption maximum 410nm). Only protein thiols that are accessible to this water-solublereagent are modified. Alternatively, the Measure-iT™ Thiol Assay Kitfrom Invitrogen can be used.

In Vivo Testing in Suitable Tumor Models.

Subcutaneous xenograft models using AR42J tumor-bearing female SCID micewill be generated. Briefly, AR42J cells (1×10⁷), will be injectedsubcutaneously into the flanks of female SCID mice. The tumors will beallowed to grow 10-12 days until 0.5-0.9 cm³ in size.

Radiolabeling of the DFO-nanoparticle by ⁸⁹Zr is expected to proceed in<15 min. at room temperature. Non-specifically bound ⁸⁹Zr will beremoved by addition of EDTA followed by a gel filtration step.

Receptor binding assays. The receptor binding assays will be performedusing ⁸⁹Zr-DFO-nanoparticles on membranes obtained from AR42J tumors.The competing ligands, natZr-DFO-Nanoparticles and natZr-DFO-octreotatewill be prepared by the reaction of high purity natural zirconiumoxalate with DFO-octreotate and DFO-Nanoparticles, respectively. Purityof the final products will be confirmed by HPLC. IC50 values will bedetermined according to previously published methods, using theMillipore MultiScreen assay system (Bedford, Mass.). Data analysis willbe performed using the programs GraFit (Erithacus Software, U.K.),LIGAND (NIH, Bethesda, Md.), and GraphPad PRISM™ (San Diego, Calif.).

In vitro assays. The AR42J cells will be harvested from monolayers withCell Dissociation Solution (Sigma Chemical Co., St. Louis, Mo.) andresuspended in fresh DMEM media at a concentration of 2×106 cells/mL. Analiquot of about 0.3 pmol of ⁸⁹Zr-DFO-nanoparticles will be added to 10mL of cells, incubated at 37° C. with continuous agitation. At 1, 5, 15,30, 45, 60 and 120 min triplicate 200-μL aliquots will be removed andplaced in ice. The cells will immediately be isolated by centrifugation,and the % uptake of the compound into the cells will be calculated.

Biodistribution. A small amount of the ⁸⁹Zr-DFO-nanoparticles (˜10 μCi,0.20 μg) will be injected intravenously into each of the mice bearingpalpable AR42J-positive tumors. The animals will be sacrificed atselected time points after injection (1, 4, 24, 48, 72 hours; n=4-5) anddesired tissues will be removed, weighed, and counted for radioactivityaccumulation. Two additional control groups will be studied at 1 hpost-injections: (A) mice injected with the native radiolabeled peptide⁸⁹Zr-DFO-octreotate (˜10 μCi, 0.20 μg), and (B) mice pre-injected with ablockade of Tyr3-octreotate (150 μg) to demonstrate receptor-mediatedaccumulation of the ⁸⁹Zr-DFO-nanoparticles. Tissues including blood,lung, liver, spleen, kidney, adrenals (STTR positive) muscle, skin, fat,heart, brain, bone, pancreas (STTR positive), small intestine, largeintestine, and AR42J tumor will be counted. The percentage injected doseper gram (% ID/g) and percentage injected dose per organ (% ID/organ)will be calculated by comparison to a weighed, counted standardsolution.

In vivo NIRF imaging. Serial imaging will be performed using theMaestro™ In Vivo Fluorescence Imaging System (CRI, Woburn, Mass.) at 0,0.5, 1, 2, 4, 6, 12, 24, 48 and 72 hrs. At 72-hr, mice will beeuthanized, and major tissues/organs dissected, weighed, and placed in6-well plates for ex-vivo imaging. Fluorescence emission will beanalyzed using regions-of-interest (ROIs) over tumor, selected tissues,and reference injectates, employing spectral unmixing algorithms toeliminate autofluorescence. Fluorescence intensities and standarddeviations (SD) will be averaged for groups of 5 animals. Dividingaverage fluorescence intensities of tissues by injectate values willpermit comparisons to be made among the various tissues/organs for eachinjected nanoparticle conjugate.

In vivo small animal PET imaging. Small animal PET imaging will beperformed on a microPET®-FOCUS™ system (Concorde Microsystems Inc,Knoxville Tenn.). Mice bearing the AR42J tumors (n=5 per group) will beanesthetized with 1-2% isoflurane, placed in a supine position, andimmobilized in a custom prepared cradle. The mice will receive 200 μCiof the ⁸⁹Zr-DFO-octreotate-nanoparticle complex via the tail vein andwill be imaged side by side. Animals will initially be imaged byacquiring multiple, successive 10-minute scans continuously from thetime of injection over a 1-hr time frame, followed by 10-min static dataacquisitions at 2, 4, 24, 48 and 72-hrs post-injection. Standard uptakevalues (SUVs) will be generated from regions of interest (ROIs) drawnover the tumor and other organs of interest. Co-registration of the PETimages will be achieved in combination with a microCAT-II camera (ImtekInc., Knoxville, Tenn.), which provides high-resolution X-ray CTanatomical images. The image registration between microCT and PET imageswill be accomplished by using a landmark registration technique andAMIRA image display software (AMIRA, TGS Inc, San Diego, Calif.). Theregistration method proceeds by rigid transformation of the microCTimages from landmarks provided by fiducials directly attached to theanimal bed.

Pharmacokinetic measurements. The biodistribution and dynamic PET datawill provide the temporal concentration of⁸⁹Zr-DFO-octreotate-nanoparticle in tissue which will allow forcharacterization of pharmacokinetic parameters of the agent.

Fluorescence microscopy and autoradiography of tissues ex vivo.Localization of nanoparticle conjugates in tissues will be performed onfrozen sections. Imaging by microPET will allow us to evaluate fully theglobal distribution in tumors and other non-target tissues. Followingthe acute stage of the imaging trial, autoradiography will also beperformed on the tumors, and this data will be correlated to both thePET imaging and histological results. Consecutive slices (˜10 μm) willbe taken, alternating slices for autoradiography and for histologicalanalysis. These sections will also be analyzed by multichannelfluorescence microscopy in the NIR channel.

Example 11 Particle Internalization Studies

The goal of this study is to evaluate the binding and internalization ofthe present nanoparticles to assess their localization in subcellularorganelles and exocytosis. This will help study the fate offunctionalized particles with different targeting moieties and attachedtherapies. For example, both diagnostic nanoparticles (e.g.,non-targeted PEG-coated versus cRGD-PEG-coated nanoparticles) andtherapeutic nanoparticles (e.g., cRGD-PEG-nanoparticles attached toiodine for radiotherapy, attached to tyrosine kinase inhibitors, orattached to chemotherapeutic drugs such as Taxol.)

Materials and Methods.

Internalization/uptake studies. Internalization assays andcolocalization studies were performed for identifying specific uptakepathways.

Melanoma cells, including human M21 and mouse B16 cells (˜2×10⁵cells/well), were plated in 8-well chamber slides (1.7 cm²/well) slidesor 24 well plates (1.9 cm²/well) with a 12 mm rounded coverglass andincubated at 37° C. overnight. To monitor targeted nanoparticleinternalization, cells were incubated with cRGD-PEG dots (0.075 mg/ml)for 3 hrs at 37° C. To remove unbound particles in the medium, cellswere rinsed twice with PBS. Confocal microscopy was performed on a Leicainverted confocal microscope (Leica TCS SP2 AOBS) equipped with a HCX PLAPO: 63x 1.2NA Water DICD objective to assess co-localization ofcRGD-PEG-dots with organelle-specific stains or antibodies. Images wereanalyzed using ImageJ software version 1.37 (NIH Image;http://rsbweb.nih.gov/ij/).

Co-localization Assays/Dye-bound markers. In order to identify endocyticvesicles involved in C dot internalization, colocalization assays inliving cells were performed using dye-bound markers. Cells werecoincubated with nanoparticles and different dyes. The dyes include: 100nM Lysotracker red for 30 min to label acidic organelles along endosomalpathway; 2 μg/mL transferrin Alexa 488 conjugate to label recycling andsorting endosomes (clathrin-dependent pathway); 1 mg/mL70 kDadextran-FITC conjugate at 37° C. for 30 min to label macropinosomes.

Co-localization/Organelle-specific antibodies. Immunocytochemistry willbe performed with known markers for Golgi and lysosomes. For Golgi,Giantin (Abcam, rabbit polyclonal, 1:2000) will be used for human cells;GM-130 (BD Pharmingen, 1 μg/ml) will be used for mouse cells. ForLysosomes, LC3B (Cell Signaling, rabbit polyclonal, 0.5 μg/ml) will beused.

For Giantin or LC3B staining, cells will be blocked for 30 minutes in10% normal goat serum/0.2% BSA in PBS. Primary antibody incubation(rabbit polyclonal anti-Giantin antibody (Abcam catalog # ab24586,1:2000 dilution) or LC3B (Cell Signaling, C#2775, 0.5 ug/ml) will bedone for 3 hours, followed by 60 minutes incubation with biotinylatedgoat anti-rabbit IgG (Vector labs, cat#:PK6101) in 1:200 dilution.Detection will be performed with Secondary Antibody Blocker, Blocker D,Streptavidin-HRP D (Ventana Medical Systems) and DAB Detection Kit(Ventana Medical Systems) according to manufacturer instructions.

For GM-130 staining, cells will be blocked for 30 min in Mouse IgGBlocking reagent (Vector Labs, Cat#: MKB-2213) in PBS. The primaryantibody incubation (monoclonal anti-GM130, from BD Pharmingen;Cat#610822, concentration 1 ug/mL) will be done for 3 hours, followed by60 minutes incubation of biotinylated mouse secondary antibody (VectorLabs, MOM Kit BMK-2202), in 1:200 dilution. Detection will be performedwith Secondary Antibody Blocker, Blocker D, Streptavidin-HRP D (VentanaMedical Systems) and DAB Detection Kit (Ventana Medical Systems)according to manufacturer instructions.

For temperature-dependent studies, nanoparticles will be incubated withcRGD-PEG-nanoparticles at 4° C., 25° C., and 37° C. to assess fractionof surface bound versus internalized particles.

For exocytosis studies, nanoparticles (0.075 mg/ml) will be incubatedfor 4 hours and chamber slides washed with PBS, followed by addition offresh media. At time intervals of 0.5, 1.0, 1.5, 2.5, 4.5, 8.0 hrs,cells will be washed, typsinized, and fluorescence signal of cells andmedia measured by fluorimetry. In dose-response studies, cells will beincubated over a range of concentrations and incubation times, andassayed using flow cytometry. In viability studies, cell viability willbe measured using a trypan blue exclusion assay before and afterincubation to assess for toxicity. In time-lapse studies, mechanism ofnanoparticle internalization in living cells will be investigated afterincubating cells with nanoparticle conjugates at different temperaturesof incubation (4° C., 25° C., and 37° C.) using an inverted confocalmicroscope over a 12-hr period at 20 min intervals.

Discussion.

cRGD-PEG-dots and PEG-dots were found to co-localize with LysotrackerRed in M21 and B16 cells suggesting uptake in the endosomal pathway(FIG. 22). Data showed that these particles strongly colocalize withtransferrin and dextran. Regardless of surface functionality and totalcharge, nanoparticles (6-7 nm in hydrodynamic diameter) studied appearedto follow the same route. Time lapse imaging in both cell typesdemonstrated internalization of functionalized nanoparticles within asmall fraction of the plated cells. Particles were eventually deliveredto vesicular structures in the perinuclear region. Colocalization assayswith Giantin (or GM-130) is not expected to show nanoparticlefluorescent signal in the Golgi.

The scope of the present invention is not limited by what has beenspecifically shown and described hereinabove. Those skilled in the artwill recognize that there are suitable alternatives to the depictedexamples of materials, configurations, constructions and dimensions.Numerous references, including patents and various publications, arecited and discussed in the description of this invention. The citationand discussion of such references is provided merely to clarify thedescription of the present invention and is not an admission that anyreference is prior art to the invention described herein. All referencescited and discussed in this specification are incorporated herein byreference in their entirety. Variations, modifications and otherimplementations of what is described herein will occur to those ofordinary skill in the art without departing from the spirit and scope ofthe invention. While certain embodiments of the present invention havebeen shown and described, it will be obvious to those skilled in the artthat changes and modifications may be made without departing from thespirit and scope of the invention. The matter set forth in the foregoingdescription and accompanying drawings is offered by way of illustrationonly and not as a limitation.

1.-75. (canceled)
 76. A composition comprising: an organic polymerattached to the nanoparticle, thereby coating the nanoparticle; and aplurality of alpha-MSH peptide ligands attached to the polymer-coatednanoparticle, wherein the nanoparticle has a diameter from 1 nm to 25 nmas measured by dynamic light scattering.
 77. The composition of claim76, wherein the plurality of alpha-MSH peptide ligands are no greaterthan twenty in number.
 78. The composition of claim 76, furthercomprising: a silica-based core; a fluorescent compound within the core;and a silica shell surrounding at least a portion of the core.
 79. Thecomposition of claim 76, wherein the organic polymer comprisespolyethylene glycol.
 80. The composition of claim 79, wherein thepolyethylene glycol is attached to a silica surface of the nanoparticlevia an amino-silane coupled to an activated ester group on the organicpolymer leading to an amide bond.
 81. The composition of claim 80,wherein the nanoparticle is coated with maleimido-terminatedpolyethylene glycol chains for attachment of the plurality of alpha-MSHpeptide ligands.
 82. The composition of claim 76, wherein the pluralityof alpha-MSH peptide ligands is no greater than ten in number.
 83. Thecomposition of claim 76, wherein the nanoparticle is coated withmaleimido-terminated polyethylene glycol chains for attachment of theplurality of alpha-MSH peptide ligands, and wherein at least onealpha-MSH peptide ligand is attached to a maleimido-terminatedpolyethylene glycol chain via a thiol group of a cysteine linker. 84.The composition of claim 76, wherein the plurality of alpha-MSH peptideligands is labeled with a radionuclide.
 85. The composition of claim 84,wherein the plurality of alpha-MSH peptide ligands is labeled with theradionuclide via a tyrosine (Y) linker.
 86. The composition of claim 76,further comprising a therapeutic agent.
 87. The composition of claim 78,wherein the fluorescent compound is Cy5.
 88. The composition of claim78, wherein the fluorescent compound is Cy5.5.
 89. The composition ofclaim 76, having a diameter from about 1 nm to about 8 nm.